Beadle was born in Wahoo, Nebraska, and graduated from the University of Nebraska in 1926; he gained his PhD from Cornell University in 1931. He then spent two years doing research in genetics under T. H. Morgan at the California Institute of Technology. Beadle was a professor at the California Institute of Technology from 1946 until 1961 and was president of the University of Chicago from 1961 until 1968. In 1937 Beadle went to Stanford University, where in 1940 he began working with Edward Tatum on the mold Neurospora. They used nutritional mutants, which were unable to synthesize certain essential dietary compounds, to determine the sequence of various metabolic pathways. Substances similar to the missing compound were added to the mutant mold cultures to find whether or not they could substitute for the lacking chemical. If the culture survived then it could be assumed that the mold could convert the substance into the chemical it needed, showing that the nutrient was likely to be a precursor of the missing chemical.

From this and similar work Beadle and Tatum concluded that the function of a gene was to control the production of a particular enzyme and that a mutation in any one gene would cause the formation of an abnormal enzyme that would be unable to catalyze a certain step in a chain of reactions. This reasoning led to the formulation of the one gene–one enzyme hypothesis, for which Beadle and Tatum received the 1958 Nobel Prize for physiology or medicine, sharing the prize with Joshua Lederberg, who had worked with Tatum on bacterial genetics.