Long was born in Danielsville, Madison County, Georgia. He received his M.D. degree at the University of Pennsylvania in 1839. After observing the same physiological effects with diethyl ether ("ether") that Humphry Davy had described for nitrous oxide in 1800, Long used ether for the first time on March 30, 1842 to remove a tumor from the neck of a patient, James M. Venable, in Jefferson, Georgia. Long subsequently removed a second tumor from Venable and used ether as an anesthetic in amputations and childbirth. The results of these trials were published in 1849 in The Southern Medical and Surgical Journal. An original copy of this publication is held in the U.S. National Library of Medicine.

Crawford Long was a member of the Demosthenian Literary Society while a student at the University of Georgia and shared a room with Alexander Stephens, Vice President of the Confederate States of America during the American Civil War. Long was a cousin of the western legend Doc Holliday.

Long died in Athens, Georgia in 1878. The Emory University-operated Crawford Long Hospital in downtown Atlanta, Georgia was named in his honor in 1931 and retained that name for 78 years. In 2009 the hospital was renamed "Emory University Hospital Midtown". The Crawford W. Long Museum in downtown Jefferson, Georgia, has been in operation since 1957. Crawford Long Middle School, in Atlanta, Georgia, was also named in his honor. A statue of Crawford Long stands in the crypt of the United States Capitol as one of the two designated monuments to represent the state of Georgia in the National Statuary Hall Collection.

Although William T.G. Morton is well-known for performing his historic demonstration of ether anesthesia on October 16, 1846 in Boston, Massachusetts, Long is now regarded as the first to have administered ether anesthesia for surgery. Morton is generally credited with the first public demonstration of ether anesthesia at the Massachusetts General Hospital, yet Long had informed several surgical colleagues who similarly administered ether in their practices. In 1854, Long requested William Crosby Dawson, a U.S. Senator, to present his claims to the attention of Congress.