Blakeslee was born at Geneseo in New York State and educated at the Wesleyan University, Connecticut, graduating in 1896. He taught science for four years before entering Harvard to do post-graduate research, gaining his PhD in 1904. In this year he discovered that the bread molds (Mucorales) exhibit heterothallism (self sterility) and spent the next two years in Germany making further investigations on the fungi.

From 1907 to 1914 Blakeslee was professor of botany at Connecticut Agricultural College. In 1915 he moved to the department of genetics at the Carnegie Institution, where he remained until 1941. In 1924 he began work on the alkaloid colchicine, which is found in the autumn crocus, and 13 years later he discovered that plants soaked in this alkaloid had multiple sets of chromosomes in their cells. Such plants, termed polyploids, often exhibit gigantism and this discovery proved of immediate use in the horticultural industry in producing giant varieties of popular ornamentals. More importantly, however, colchicine often converts sterile hybrids into fertile polyploids and is therefore an invaluable tool in crop-breeding research.

Other contributions made by Blakeslee to plant genetics include his study of inheritance in the jimson weed and his research on embryo culture as a method of growing hybrid embryos that would abort if left on the parent plant.