Longley's most notable scientific work focused on the coloration and patterning of tropical reef fish. He conducted this research with the support of the Carnegie Institution's Dry Tortugas Laboratory in Washington, D.C., where he served as director from 1922 to 1937. His investigations also encompassed the distribution and the evolution of the tropical reef fish.
Longley's passion for plant life extended beyond marine biology. He conducted extensive botanical studies in various locations, including Hawaii, Samoa, the Tortugas islands, and the Pacific region. His research also involved examining plant specimens housed in European and American museums.[2]