Hickok was born in Troy Grove, Illinois on May 27, 1837. He left his father's farm in 1855 to be a stage coach driver on the Santa Fe and Oregon Trails. His gunfighting skills led to his nickname. In 1857, he claimed a 160 acre (exactly 1/4 mi²=about 0.65 km²) tract of land in Johnson County, Kansas (in what is now the city of Lenexa) where he became the first constable of Monticello Township, Kansas .

In 1861, he became a town constable in Nebraska. He became well-known for single-handedly capturing the McCanles gang at Rock Creek Station through the use of force. On several other occasions, Hickok confronted and killed several men while fighting alone. His famous statement to Phil Coe, who supposedly stated he could "kill a crow on the wing," (flying) is one of the Old West's most famous sayings, and showed that Hickok was certainly a cool customer in a fight. He answered Coe by sneering, "Did the crow have a pistol? Was he shooting back? I will be." Hickok later killed Coe. Whether or not Coe had actually made the crow brag, and Hickok answered as reported, it certainly personified the reputation Wild Bill accrued.

After the American Civil War, Hickok became an Army scout and a professional gambler, and served as a United States Marshal. In 1867, his fame increased from an interview by Henry Morton Stanley. Hickok's killing of Whistler the Peacemaker with a long-range rifle shot had influence in preventing the Sioux from uniting to resist the settler incursions into the Black Hills. While Sherriff/City Marshal of Hays, Kansas on July 17, 1870, he was involved in a gunfight with soldiers of the 7th US Cavalry wounding one and mortally wounding one. In 1871, Hickok became marshal of Abilene, Kansas. His encounter there with John Wesley Hardin resulted in the latter fleeing the town after Wild Bill managed to disarm him. On Oct 5, 1871 he accidentally shot and killed Abilene Special Deputy Marshall Mike Williams . In 1873-1874, Hickok joined Buffalo Bill Cody and Texas Jack Omohundro in a touring stage play titled Scouts of the Plains, the forerunner to Cody's Wild West shows. There, he befriended "Calamity" Jane Cannary-Burke, who was later to claim a romantic relationship which appears dubious as Hickok was newly married and greatly enamored of his wife. He was fired from the show due to drunkenness. The two were to meet again in Charlie Utter's 1876 wagon train from Colorado to Deadwood, South Dakota, where the three of them remained close friends.

It is difficult to separate the truth from fiction about Hickok, the first "dime novel" hero of the western era. Hickok himself told the writers with great seriousness that he had killed over 100 men. While this number is doubtful, there is no doubt that Hickok was a fearless and deadly fighting man, equally at home with a rifle, revolver, or knife.

Hickok invented the concept of "posting" men out of town. He would put a list on what was called the "dead man's tree" (men had been lynched on it) while constable of Monticello Township. If they were not gone by sundown of that day, Hickok proclaimed he would shoot them on sight the following day. Few stayed around to find out if he was serious.

On August 2, 1876, while playing poker at Nuttal & Mann's "Saloon No. 10" in Deadwood (then part of the Dakota Territory but on Indian land), Hickok could not find an empty seat in the corner, where he always sat in order to protect himself against sneak attacks from behind, and instead sat with his back to the door; unfortunately, his previous caution proved wise, as he was shot in the back of the head with a double-action .45 caliber revolver by Jack McCall. The motive for the killing is still debated. (McCall may have been paid for the deed, it may have just been the result of a recent dispute, or McCall may, in a drunken rage, have become enraged over what he perceived as a condescending offer from Hickok to let him have enough money for breakfast after he had lost all his money playing poker the previous day.) McCall claimed at the resulting two-hour trial by a motley group of assembled miners and businessmen that he was avenging Hickok's earlier slaying of his brother and was acquitted, resulting in the Black Hills Pioneer editorializing:

"Should it ever be our misfortune to kill a man ... we would simply ask that our trial may take place in some of the mining camps of these hills." McCall was subsequently rearrested after bragging about his deed, and a new trial was held. This was not double jeopardy because Deadwood at the time was an illegal city due to several laws that made it illegal to settle on Indian land, although many people did anyway. The new trial was held in American territory, in Yankton, South Dakota. Hickok's brother, Lorenzo Butler Hickok, traveled from Illinois to attend the retrial. This time McCall was found guilty and hanged. After his execution it was determined that McCall had never even had a brother. The saloon proprietor claimed that, at the time of his death, Hickok held a pair of aces and a pair of eights, with all cards black, and this has since been called a "dead man's hand".

Utter claimed the body, and placed a notice in the local newspaper, the Black Hills Pioneer, which read:

"Died in Deadwood, Black Hills, August 2, 1876, from the effects of a pistol shot, J. B. Hickok (Wild Bill) formerly of Cheyenne, Wyoming. Funeral services will be held at Charlie Utter's Camp, on Thursday afternoon, August 3, 1876, at 3 o'clock, P. M. All are respectfully invited to attend." Almost the entire town attended the funeral, and Utter had Hickok buried with a wooden grave marker reading:

"Wild Bill, J. B. Hickok killed by the assassin Jack McCall in Deadwood, Black Hills, August 2d, 1876. Pard, we will meet again in the happy hunting ground to part no more. Good bye, Colorado Charlie, C. H. Utter." At the urging of Calamity Jane, Utter in 1879 had Hickok reinterred in a ten foot square plot at the Mount Moriah Cemetery, surrounded by a cast-iron fence with an American flag flying nearby. A monument has since been built there. In accordance with her dying wish, Calamity Jane was buried next to him.

Shortly before Hickok's death, he wrote a letter to his new wife, which in retrospect seems eerily prescient:

"Agnes Darling, if such should be we never meet again, while firing my last shot, I will gently breathe the name of my wife - Agnes - and with wishes even for my enemies I will make the plunge and try to swim to the other shore". A week before Hickok's death he wrote a letter to his wife, Agnes, containing: "My dearly beloved if I am to die today and never see the sweet face of you i want you to know that I am no great man and am lucky to have such a woman as you."