Flemming was born at Sachsenberg, now in Germany, and graduated in medicine from the University of Rostock in 1868. However, after a short period working in a hospital, he turned to physiology and became assistant to Willy Kuhne at the Institute of Physiology in Amsterdam. After serving as a physician in the Franco-Prussian War he held professional posts at Prague (1873) and Kiel (1876).

By making use of the newly synthesized aniline dyes Flemming was able to discern the threadlike structures in the cell nucleus, which Heinrich Waldeyer was later to term chromosomes. The new staining techniques made it possible for Flemming to follow in far greater detail the process of cell division, which he named ‘mitosis’ from the Greek for thread. Most importantly, Flemming detailed the fundamental process of mitosis, that is, the splitting of the chromosomes along their lengths into two identical halves. These results were published in the seminal book Zell-substanz, Kern und Zelltheilung (1882; Cytoplasm, Nucleus and Cell Division). It was another 20 years before the significance of Flemming's work was truly realized with the rediscovery of Gregor Mendel's rules of heredity.