Alfred Henry Sturtevant was born in Jacksonville, Illinois. Sturtevant was always interested in inheritance and genetics. One of Sturtevant's earliest publications was a pedigree analysis of horses owned by his father. In 1909, while an undergraduate at Columbia University, Sturtevant attended a lecture given by Thomas Hunt Morgan. It was one of the few undergraduate classes that Morgan ever taught. Morgan's passion for science and discovery interested Sturtevant so much that he approached Morgan about working for him. Sturtevant became one of Morgan's first students in the "Fly room" to work on Drosophila melanogaster.

For his Ph.D. thesis, Sturtevant published the world's first genetic map. The idea of gene linkage came to him in a flash one night. He and the other members of Morgan's lab had been discussing a paper on the coat color of rabbits. Sturtevant realized that genes were linked in a series, and data as to how these genes were linked could be deduced by building the "right" Drosophila mutant. Sturtevant stayed up most of one night working out the details of linkage analysis instead of doing his undergraduate homework.

In 1928, Sturtevant, along with Thomas Hunt Morgan and Calvin Bridges, moved to the California Institute of Technology. Sturtevant was a Professor of Biology at Cal Tech until 1951.