Efforts to preserve the Rookery Bay estuary were initiated in 1964, when developmental pressures were directed toward this relatively undisturbed estuary. A proposed road would have allowed access to the area and facilitated residential development. However, local opposition resulted in a site recommendation for preservation. Instrumental in this action were the newly founded Collier County Conservancy, now the Conservancy of Southwest Florida, the National Audubon Society, and The Nature Conservancy. From 1964 through 1974, over 3,700 acres of lands associated with Rookery Bay were primarily acquired through the efforts of these three organizations. The title for most of these wetlands was vested in the National Audubon Society, and the area was designated as an Audubon Wildlife Sanctuary.

In order to provide adequate protection for the Rookery Bay ecosystem and establish a long-term source of operational funds, the three groups requested that the State of Florida apply to the National Oceanic and Atmospheric Administration (NOAA) for National Estuarine Research Reserve (NERR) status for Rookery Bay. As a condition of the designation process, these parties signed an agreement with the State in 1977 that leased Audubon's holdings around Rookery Bay to the State of Florida for 99 years.

In September 1978, Rookery Bay was formally designated a NERR. Since the designation of Rookery Bay National Estuarine Research Reserve has constructed and operates an on-site headquarters, a two-story visitor center, research laboratories, two field research stations and dormitories, a boat dock and maintenance/fleet support facilities.