Lewis graduated from E. L. Meyers High School. He received a BA in Biostatistics from the University of Minnesota in 1939, where he worked on Drosophila melanogaster in the lab of C.P. Oliver. In 1942 Lewis received a Ph.D. from California Institute of Technology (Caltech), working under the guidance of Alfred Sturtevant.

After serving as a Meteorologist in the U.S. Air Force in World War II, Lewis joined the Caltech faculty in 1946 as an instructor. In 1956 he was appointed Professor of Biology, and in 1966 the Thomas Hunt Morgan Professor of Biology.

During the 1950s, Lewis studied the effects of radiation from X-rays, nuclear fallout and other sources as possible causes of cancer. He reviewed medical records from survivors of the atomic bombings of Hiroshima and Nagasaki, as well as radiologists and patients exposed to X-rays. Lewis concluded that "health risks from radiation had been underestimated". Lewis published articles in Science and other journals and made a presentation to a Congressional committee on atomic Energy in 1957.

The issue of linearity versus threshold re-entered the debate on nuclear fallout in 1962, when Ernest Sternglass, a Pittsburgh Physicist, argued that the linearity thesis was confirmed by the research of Alice Stewart. (See also John Gofman )

Lewis received numerous awards and honours during his career including the Thomas Hunt Morgan Medal in 1983, the Louisa Gross Horwitz Prize in 1992, the Albert Lasker Award for Basic Medical Research in 1991 and the Nobel Prize in Medicine in 1995. He was elected a Foreign Member of the Royal Society (ForMemRS) in 1989. He was also awarded the Gairdner Foundation International award in 1987, the Wolf Foundation prize in Medicine in 1989, the Rosenstiel award in 1990 and the National Medal of Science in 1990.

On November 20, 2001 Lewis was interviewed by Elliot Meyerowitz in the Kerckhoff Library at the California Institute of Technology, Pasadena, California. This interview was released on DVD in 2004 as "Conversations in Genetics: Volume 1, No. 3 - Edward B. Lewis; An Oral History of Our Intellectual Heritage in Genetics" 67 min; Producer Rochelle Easton Esposito; The Genetics Society of America.

His Nobel Prize–winning studies with Drosophila, (including the discovery of the Drosophila Bithorax complex of homeotic genes, and elucidation of its function), founded the field of evolutionary developmental biology and laid the groundwork for our current understanding of the universal, evolutionarily conserved strategies controlling animal development. He is credited with development of the complementation test. His key publications in the fields of genetics, developmental biology, radiation and cancer are presented in the book Genes, Development and Cancer, which was released in 2004.