The 23-year-old has agreed a two-and-a-half-year contract with an option for a further 12 months.
"He's left-footed, which gives us good balance. We've been looking for a left-footer for a while," boss Nathan Jones told the Luton website.
Gambin has six caps, but was suspended for Malta's 2-0 defeat by England at Wembley last October.
He made 23 appearances for Barnet this season, scoring four goals, the last in a 3-1 defeat at Luton on New Year's Eve.
The Hatters are currently sixth in League Two, six points off the automatic promotion places.
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Luton Town have signed Malta international winger Luke Gambin from Barnet for an undisclosed fee.