From 1868 to 1870, Ross starred for the Rockford Forest Citys, along with Albert Spalding, attaining professional status in the second year. When the National Association was formed in 1871, Harry Wright signed both men to his new team in Boston. Barnes' major league career thus started when he was only 21. He split time between second base and shortstop for the Boston Red Stockings of the new National Association. Barnes led the league with 66 runs scored and 91 total bases, finishing second in batting average at .401.

In 1872 he led the Association with a .432 batting average, a .585 slugging percentage, 99 base hits, 134 total bases, and 28 doubles. The Red Stockings began a four-year dominance of the Association, with Barnes a key player each year.

Barnes again led the Association in 1873, hitting .425, as well as leading in on base percentage (.456),slugging percentage (.584), base hits (137), runs scored (125), total bases (188), doubles (29), bases on balls (28), and stolen bases (13).

His .340 BA in 1874 was only good enough for eighth in the league, while his .364 was good for second in 1875, while leading again in runs scored (115), base hits (143) and on base percentage (.375).

Before the 1875 season ended, Barnes and four other Boston players signed contracts with the Chicago White Stockings. When word leaked out in Boston before the end of the season, Barnes and his teammates were reviled by Boston fans, being called "seceders", a strong epithet just a decade after the Civil War. It was likely that the National Association would void the signing, but Chicago owner William Hulbert preempted the move by forming the National League, and causing the NA to disband.

Barnes' new team finished first in the NL's first season with a 55-12 record, while Boston fell to fourth. Ross led the National League batting (.429), on base percentage (.462), slugging (.562), runs (126), hits (138), bases (190), doubles (21), triples (14), and walks (20). For those first six years of major league play, he had hit .397. However, it was to be his last dominant season.

In 1877, Ross fell ill with what was then only described as an "ague" (fever), played only 22 games, and did not play well when he was in the lineup. The illness robbed him of much of his strength and agility, and shortened his career. While many baseball histories originally blamed the change in rules that outlawed the "fair-foul" hit, of which Barnes was an acknowledged master, his illness has become a more widely accepted explanation for his loss of productivity.

The remainder of his career was an effort to return to glory ending in mediocrity. He played for the Tecumseh team in the International Association (arguably baseball's first minor league) in 1878, returned to the National League with the Cincinnati club in 1879, sat out all of 1880, and finished his professional career in 1881, playing his last season in Boston, site of his former glory. After 1876, he never hit better than .272, and his other totals were barely half of those from his glory days. He retired at age 31. He finished his career with 859 hits, 698 runs, and a .359 average, in only 499 games played and 2392 at bats. His 1.4 runs per game played remains the best of all time.

Barnes has been rated as the best player of the National Association, and during his peak, from 1871 to 1876, he was a dominant offensive force. His skill at the fair-foul bunt caused rule changes, and his defensive abilities were highly regarded. A teammate of multiple members of the Baseball Hall of Fame, he was the most valuable batter. He also has the distinction of having hit the first home run in National League history, on May 2, 1876.

A lifelong bachelor, Barnes held a variety of white-collar jobs in the Chicago area after his baseball career ended until his death from heart disease in 1915.