Enders was born in West Hartford, Connecticut, and was educated at the Noah Webster School at Hartford and St. Paul's School in Concord, New Hampshire. He then attended Yale University for a short time before entering the United States Army Air Corps in 1918. After returning from war he graduated from Yale, where he was a member of Scroll and Key as well as Delta Kappa Epsilon, and went on to become a businessman in real estate in 1922. He tried his hand at several careers before choosing to work in the biological field studying infectious diseases, gaining a Ph.D. at Harvard in 1930.

In 1954, while working at Children's Hospital Boston, Enders, Thomas Huckle Weller, and Frederick Chapman Robbins were awarded the Nobel Prize in Physiology or Medicine "for their discovery of the ability of poliomyelitis viruses to grow in cultures of various types of tissue". This work was the first to show that viruses of this type could be grown and manipulated outside of the body. It was this technique dubbed the Enders-Weller-Robbins method that Jonas Salk used to develop the polio vaccine in 1952. A large-scale test of the vaccine proved successful in 1954, relying on Enders method of virus culture. Because of the significant advance to vaccine research the cell culture system allowed, many professionals in the field still regard Enders', Weller's and Robbins' work as substantial and why Enders has received the title of the "Father of Modern Vaccines".

Enders died in 1985 in Waterford, Connecticut, aged 88.