As a child in Illinois, Hickok worked on neighbouring farms and helped his father in assisting escaped slaves. He left home in 1856 to farm in Kansas and there became involved in the Free State (antislavery) movement. He later served as a village constable in Monticello, Kansas. While working as a teamster in 1861, he killed Dave McCanles at Rock Creek (Nebraska Territory), and legends about him probably began in the exaggerated tales of his role in this gunfight.

During the American Civil War Hickok worked for the Union as a teamster, scout, and spy. After the war he was appointed deputy U.S. marshal, and he later became a scout for the army. Hickok is remembered particularly for his services in Kansas as sheriff of Hays City and marshal of Abilene, where his ironhanded rule helped to tame two of the most lawless towns on the frontier. In 1872 Hickok emceed an unsuccessful Wild West show, and in 1873–74 he performed with Buffalo Bill (Cody, William F.)'s theatrical troupe.

In 1876 Hickok married a widow, Mrs. Agnes Lake Thatcher, née Mersman, but he soon left her (in Cincinnati) to visit the goldfields of the Black Hills in the Dakota Territory. It was there, at a poker table in Nuttall & Mann's No. 10 saloon in Deadwood, that Hickok was shot dead by a drunken stranger, Jack McCall. The cards Hickok was holding—a pair of black aces and a pair of black eights plus an unknown fifth card—became known as the dead man's hand. McCall's motive was never learned; he was tried, convicted of murder, and hanged on March 1, 1877.