Stanley obtained his doctorate from the University of Illinois in 1929. He worked from 1932 to 1948 at the Rockefeller Institute for Medical Research (now Rockefeller University) facilities in Princeton, N.J. In 1935 Stanley crystallized tobacco mosaic virus (TMV, the causative agent of a plant disease) and showed that it is a rod-shaped aggregate of protein and nucleic acid molecules. His work enabled other scientists, utilizing methods of X-ray diffraction, to ascertain unambiguously the precise molecular structures and the modes of propagation of several viruses.

While a professor of biochemistry and director of the laboratory for virus research at the University of California, Berkeley (1948–71), Stanley studied influenza viruses, for which he developed a preventive vaccine.