The 23-year-old Germany international, who has three years remaining on his contract, returns to the Bundesliga 19 months after joining Spurs from Schalke.
Holtby spent time on loan at Fulham last season had been linked with another temporary move to a Premier League rival, with Everton at one stage thought to be interested.
Hamburg's director of sport Dietmar Beiersdorfer said: "Lewis has great passing ability and he is going to do us good with his creativity.
"He's just 23 but he already has a great deal of experience in the Bundesliga, the Premier League as well as in Europe and he should now bring that experience here with him."
Two other players on their way out of White Hart Lane are youngsters Tomislav Gomelt and Souleymane Coulibaly, who have both moved to Bari.
Gomelt, 19, joins the Italian Serie B side on a season-long loan while Coulibaly, also 19, has signed permanently.
Striker Jonathan Obika has also left Spurs after signing for Swindon Town for an undisclosed fee.The 23-year-old, who made five appearances for the Robins during a loan spell in the 2010/11 season, has put pen to paper on a two-year deal at the County Ground