The seven-time German champions have never been relegated from the Bundesliga since its formation in 1963.
However, a desperate campaign has yielded just seven wins - with Mirko Slomka having replaced Bert van Marwijk as coach in February.
Despite ending the season with five consecutive wins, Hamburg managed to beat the automatic drop, as fellow strugglers Nuremberg and Eintracht Braunschweig were both well beaten by Schalke and Hoffenheim respectively on the final day.
Slomka's men now face a two-legged play-off against 2.Bundesliga side Greuther Furth, who finished third in the second tier and will be hoping to reach the Bundesliga for the second time in their history, having been relegated 12 months ago.
Ilir Azemi's return of 14 goals in 28 games has been crucial for Greuther, but Hamburg captain Rafael van der Vaart is confident his side will finally seize the chance to stay up.
Speaking after Saturday's season-ending defeat against Mainz, the Dutch star said "We still have the chance to stay up and we plan to take it.
"We have to take the positives and show that we are a Bundesliga team. The support we had here was once again incredible. Our fans were the man of the match."
Hamburg, one of Germany's oldest and most prestigious clubs, have won three DFB-Pokal crowns, one European Cup and one European Cup Winner's Cup as well as their national titles.
Despite this illustrious past, they have declined in recent seasons - finishing outside the top six in each of the last five years - and relegation from the Bundesliga would be a huge blow to a club with one of the largest fanbases in Germany.
Top scorer Pierre-Michel Lasogga, who is on loan from Bundesliga rivals Hertha Berlin has 13 league goals this season added: "We will do everything possible to stay up knowing our fans will be right behind us."
The 22 year-old, whose form has earned international recognition and reported interest from abroad, had missed Hamburg's previous five games prior to the weekend due to injury, but was on target again on Saturday.
Hamburg face Greuther in what Slomka has called "a must win game" at the Imtech Arena on Thursday, with the second leg being played on Sunday.
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