The defeat comes five days after Ghana lost 2-0 to Ivory Coast in Bouake, leaving Carlos Queiroz’s side bottom of Group C and facing an early battle to keep their qualification hopes alive.
It was a slow start from both sides, but the game came to life approaching the half-hour mark when Ghana were awarded a penalty after Jerome Opoku was adjudged to have been fouled in the box following a corner.
Ernest Nuamah stepped up and calmly converted from the spot to give Ghana the lead.
The Black Stars doubled their advantage six minutes later when Felix Afena-Gyan was brought down on the edge of the penalty area.
Afena-Gyan took responsibility for the resulting free-kick and produced a fine finish to put Ghana 2-0 ahead.
The Gambia, however, were handed a route back into the contest four minutes later.
Gideon Mensah was have bundled Joseph Ceesay over inside the box, giving the Scorpions a penalty.
Mohammed Badamosi stepped up and sent Ghana goalkeeper Benjamin Asare the wrong way from 12 yards to reduce the deficit.
The visitors completed their comeback just three minutes before half-time.
A long throw was flicked on by Yaya Bojang into the path of captain Omar Colley, who kept his composure to finish and send the two teams into the break level at 2-2.
Queiroz responded at half-time with two changes, bringing on Ibrahim Sulemana and Benjamin Tetteh for Brandon Thomas-Asante and Kwasi Sibo.
Ghana started the second half strongly and pushed forward in search of a third goal, but The Gambia proved dangerous on the counter.
Against the run of play, the Scorpions took the lead when a long ball found Badamosi, who flicked it on.
Mensah failed to clear the danger and second-half substitute Ebrima Colley pounced on the loose ball before finishing past Asare to make it 3-2.
Ghana continued to push for an equaliser but were unable to find a way through the Gambian defence.
The visitors then put the game beyond Ghana when Ebrima Colley scored his second of the night, heading a Sanyang cross into an empty net to complete a memorable victory for The Gambia.
The result leaves Ghana without a point from their opening two Group C matches after defeats to Ivory Coast and The Gambia.
The Scorpions, meanwhile, have six points from their opening two games after also beating Somalia in their first qualifier.
Ghana therefore face an early uphill battle in their bid to qualify for the 2027 AFCON after missing the 2025 tournament in Morocco.
The Black Stars will now turn their attention to their remaining Group C fixtures, with matches against Somalia, Ivory Coast and The Gambia still to come.
