"Christian Traesch cannot take part in the World Cup in South Africa," the DFB said on their website.
The loss of the 22-year-old further limited Germany's options in midfield after injured captain Michael Ballack was ruled out of June 11-July 11 tournament in South Africa.
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First-choice goalkeeper Rene Adler was not even included in the preliminary 27-man squad due to a rib injury.
Traesch twisted his right ankle in the eighth minute of Germany's 4-0 win over FC South Tirol and was taken to hospital for a scan which revealed damage to his ankle.
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"This is a real shame for him, for such a thing to happen at this stage," coach Joachim Low said.
The 22-year-old VfB Stuttgart player has won two international caps and was a candidate to replace Ballack in midfield, who was ruled out of the World Cup after being injured in the English FA Cup final 10 days ago.
The injuries have narrowed down coach Joachim Loew's preliminary squad to just 25 players. He must announce a final 23-man squad by June 1.
Germany have been drawn with Serbia, Australia and Ghana in the group stage.
