Ayala, who was born in Madrid, began his higher education there at the University of Madrid, moved to America in 1961, and obtained his PhD from Columbia in 1964. He worked initially at Rockefeller before joining the Davis campus of the University of California in 1971, where he was later appointed professor of genetics in 1974. He was professor of biology at the University of California, Irving, from 1987 to 1989, when he became Donald Bren Professor of Biological Sciences.

Ayala has worked extensively in the field of molecular evolution. He has also sought to measure genetic variation in natural populations, rates of evolution, and the amount of genetic change needed to produce new species. Many of his results were published in his Molecular Evolution (1976) and in a work he coauthored in 1977 entitled Evolution. He has written a number of other books including Molecular Genetics (1984).