Construction of the airport began in April 1940 as a Works Project Administration project. In 1941 initial construction was completed. Upon conclusion of the construction by the United States Army Corps of Engineers, the facility was known as the Alachua Army Airfield and was used by the Army Air Corps and the Army Air Forces.

The airfield was declared surplus in September 1945 and turned over to the Army Corps of Engineers on October 1, 1946. The War Assets Administration deeded the facility to the city of Gainesville in 1948 as a civil airport. The field was known as John R. Alison Airport or Gainesville Municipal Airport; the city operated, maintained, and improved the airport, which was renamed the Gainesville Regional Airport in October 1977. The airline terminal was dedicated to John R. Alison in 1979.

Eastern Airlines served Gainesville beginning in the 1950s, with flights to Tallahassee, Jacksonville and Ocala, eventually offering nonstop flights to Atlanta and Miami by the 1970s. Eastern served Gainesville until its bankruptcy in 1991 and was consistently a profitable destination for the company. Air Florida served Gainesville in the late 1970s and early 1980s, with flights to Miami, Tampa and Ocala.

In 1986 the State Legislature passed a bill that established the Airport as the Gainesville-Alachua County Regional Airport Authority. The Airport Authority has nine board members, five selected by the City of Gainesville, three by the Governor and one by Alachua County, and continues to oversee the Airport.

On October 31, 2004 Gainesville Regional Airport hosted Air Force One during George W. Bush's re-election campaign. 17,000 people attended the event. They hosted Air Force Two in 2023 after Hurricane Idalia.

In recent years the Airport has substantially completed several projects: the refurbishment of its primary runway (11/29), piping of an open ditch parallel to that runway, and completion of two phases of the Terminal Renovation project. Three passenger boarding bridges have been installed.

Eclipse Aviation, maker of the Eclipse 500, operated its first factory service center in Gainesville until the company declared bankruptcy in 2009. In 2012 Silver Airways moved its maintenance facilities from Ft. Lauderdale into the existing Eclipse Aviation facility. Silver provided commercial airline service to several destinations around Florida and Bahamas at the time. In April 2015, Silver Airways moved its maintenance to Orlando International. However, in 2023, Silver Airways announced they would return to Gainesville with service to Fort Lauderdale–Hollywood International Airport. But after about 1 year of service to Gainesville, Silver announced in August 2024 that they would shut down their service due to low demand and a 28% on-time rate.

The airport's control tower operates from 6:45a to 10:30p daily. After the death of musician Tom Petty, a Gainesville native, on October 2, 2017, a change.org petition was started to have the airport renamed "Tom Petty - Gainesville Regional Airport", although as of 2025 the petition is no longer up and has been canceled.

In 2021, Gainesville Regional Airport added a 15,200 square-foot expansion onto their terminal. The new expansion adds a mother lactation room, a pet relief area, two new gates, hundreds of new seats with charging ports, a departure and arrival board, and an area for a food/store vendor. In addition to the expansion, the existing terminal was given a renovation. The project cost $16 million, funded mostly by a $12 million grant from the FAA.

In February 2024, the airport broke ground on a new four story 418-space parking garage to alleviate parking concerns. The garage will also house new ride-sharing pickup points and bus service and is expected to open by July 2025.

In September 2024, the Gainesville Regional Airport announced an agreement to install an electric charging station that can charge electric aircraft.

The airport served a record 578,000 passengers in 2024.