Best was born Jules Guy in Powderly, Kentucky in 1926. After his mother died in 1929, he was sent to live in an orphanage. He was later adopted by Armen and Essa Best and went to live with them in Corydon, Indiana, a suburb of Louisville.

He began his acting career with an uncredited role in the 1950 western One Way Street. Best would portray a wide variety of characters in a wide spectrum of film genres. Some of his more notable roles include Jason Brown in the 1955 historical drama Seven Angry Men, Kit Caswell in the 1958 western Cole Younger, Gunfighter, Dr. Ben Mizer in the 1966 comedy Three on a Couch, and the craven Dewey Barksdale in the 1976 drama Ode to Billy Joe.

He has also appeared over 600 times in numerous televisions shows including Bonanza, Gunsmoke, Have Gun, Will Travel, The Twilight Zone, The Mod Squad, The Andy Griffith Show, Perry Mason, The Fugitive, and In the Heat of the Night. Fans of the Andy Griffith Show will remember Best for his portrayal of the young guitar player Jim Lindsey.

The Dukes of Hazzard role was Best's most visible. He later revealed that the caricature-like persona of Sheriff Rosco P. Coltrane was developed from a voice he used when he would play with his young children.

After Dukes, Best continued to act. He later moved to Florida and taught at the University of Central Florida. Now semi-retired, Best runs a production company and takes occasional acting roles. He has also been reunited with several of his blood relatives including cousins Phil and Don Everly of the Everly Brothers. He has earned a name for himself as a talented artist and painter.

A highly respected acting coach, he taught drama and acting techniques for over 25 years in Los Angeles. His acting school listed some of the top names in Hollywood as pupils. He also served as artist-in-residence and taught drama at the University of Mississippi for two years prior to his stint on The Dukes of Hazzard.