Born in Southampton, New York State, Griffin was educated at Harvard where he obtained his PhD in 1942. He spent the war applying physiological principles to the design of such military equipment as cold-weather clothing and headphones. After the war he worked initially at Cornell but returned to Harvard in 1953 as professor of biology, a position he held until his retirement in 1986.

Bat navigation had been first studied by Lazzaro Spallanzani in 1793 when he noted that blinded bats were as efficient in catching insects on the wing as sighted bats. He also noted that impairment of their hearing produced disorientation. How, Griffin asked in 1938, could ears replace eyes in flight guidance? He was fortunate in that he had a colleague in the physics laboratory, G. W. Pierce, with an interest in high-frequency sound who was willing to use his specialized equipment on bats. In this way they soon found that bats were issuing sounds, in the case of the horseshoe bat, with frequencies between 60 and 120 kilohertz; the limit of human hearing is between 15 and 20 kilohertz.

That the bats were operating by echolocation was demonstrated when either their mouths or ears were covered. In either case they would collide in a dark room with anything in their path, even the room's walls. But, when operating freely, they found that the small brown bat, Myotis lucifugus, could detect in the dark and fly through a screen with wires no more than 24 centimeters apart. Only when the wires were reduced to a diameter of less than 0.07 millimeter, about the size of a human hair, did their detection system break down. Griffin went on to explore in greater detail the nature of bat sonar systems, describing his work in his Listening in the Dark (New Haven, 1958), and the more popular account Echoes of Bats and Men (London, 1960).

Griffin has also worked extensively on the problem of bird navigation. Early work inevitably consisted of homing experiments. But from these no more can be established than the percentage of birds arriving safely, and the time taken. They did, however, establish that the number of returns decreased with distance in a way that suggested that birds navigate by identifying local landmarks. To discover more about the process Griffin taught himself to fly and in the late 1940s spent many hours in a Piper Cub observing the flight paths of gannets and gulls. His observations tended to support the idea that homing from unfamiliar territory was not accurately directed, but succeeded eventually because of exploratory flights.

While this was perhaps a reasonable account of the behavior of gannets and gulls, later work by others showed that in the case of homing pigeons, shearwaters, and starlings there did exist a highly developed ability to select a particular direction. Griffin described his own work and the work of others in his Bird Migration (London, 1965).