Enders was born in West Hartford, Connecticut. His father, John Ostrom Enders, was CEO of the Hartford National Bank and left him a fortune of $19 million upon his death. He attended the Noah Webster School in Hartford, and St. Paul's School in Concord, New Hampshire. After attending Yale University a short time, he joined the United States Army Air Corps in 1918 as a FLIGHT instructor and a lieutenant.
After returning from World War I, he graduated from Yale, where he was a member of Scroll and Key as well as Delta Kappa Epsilon. He went into real estate in 1922, and tried several careers before choosing the biomedical field with a focus on infectious diseases, gaining a Ph.D. at Harvard in 1930. He later joined the faculty at Children's Hospital Boston.
In 1949, Enders, Thomas Huckle Weller, and Frederick Chapman Robbins reported successful in vitro culture of an animal virus—poliovirus. The three received the 1954 Nobel Prize in Physiology or Medicine "for their discovery of the ability of polioviruses to grow in cultures of various types of tissue".
Meanwhile, Jonas Salk applied the Enders-Weller-Robbins technique to produce large quantities of poliovirus, and then developed a polio vaccine in 1952. Upon the 1954 polio vaccine field trial, whose success Salk announced on the radio, Salk became a public hero but failed to credit the many other researchers that his effort rode upon, and was somewhat shunned by America's scientific establishment.
In 1954, Enders and Peebles isolated measlesvirus from an 11-year-old boy, David Edmonston. Disappointed by polio vaccine's development and involvement in some cases of polio and death—what Enders attributed to Salk's technique—Enders began development of measles vaccine. In October 1960, an Enders team began trials on 1,500 mentally retarded children in New York City and on 4,000 children in Nigeria.
On September 17, 1961, The New York Times announced the measles vaccine effective. Refusing credit for only himself, Enders stressed the collaborative nature of the effort. In 1963, Pfizer introduced a deactivated measles vaccine, and Merck & Co introduced an attenuated measles vaccine.
Enders died in 1985 in Waterford, Connecticut, aged 88, holding honorary doctoral degrees from 13 universities.