Last year's Norris Trophy winner as top defenceman will look to help the struggling Senators clinch a playoff spot.
Karlsson took to Twitter to express his happiness: "BOOM.! FINALLY.! Back in the line up tonight."
Karlsson's presence, coupled with a recent return of Jared Cowen from a season-long hip injury, gives Ottawa a full complement of the defencemen who returned to the club from the 2011-12 season.
Senators general manager Bryan Murray had said in February that the Swedish blue-liner was effectively done for the season after suffering a 70 per cent tear of the tendon.
Karlsson began practising with his teammates last week.
He suffered the injury when his left leg was struck by the skate blade of Pittsburgh forward Matt Cooke. Team owner Eugene Melnyk later said he believed Cooke's action was intentional, which the Penguins player denied.
Karlsson won the Norris last year in just his third season, recording 19 goals and 59 assists.