Canaries skipper Holt, the club's leading scorer with 30 goals last season, made a brief appearance from the bench following a thigh injury as Norwich lost their opening game of the Championship season to Watford last Friday.
But having replaced the ineligible Simeon Jackson - an agreement made after his switch from Gillingham to Carrow Road this summer - Holt was soon back in the swing of things on Tuesday night.
After David Fox had hit a post for City, Gillingham took the lead against the run of play in the 12th minute with Chris Palmer taking advantage of a slip by defender Michael Nelson to beat John Ruddy.
Gills nearly doubled their advantage in the 23rd minute when Adebayo Akinfenwa thumped a header from Danny Spiller's free-kick against the bar.
Norwich capitalised on the let-off by levelling three minutes later. Holt played in strike-partner Chris Martin who avoided the presence of a defender to beat Alan Julian.
Within six minutes, the Canaries had turned things around as Holt headed home Russell Martin's cross from 12 yards.
City came close to stretching their lead seven minutes before half-time, Chris Martin seeing his curling 25-yard free-kick hit the crossbar.
But 10 minutes after the restart Holt netted his second of the night, finishing off a sweeping move which resulted in Korey Smith's cross being clinically dispatched.
Chris Martin claimed his second of the night in injury time, collecting a pass from substitute Oli Johnson before rounding Julian and tapping into an empty net.