The 43-year-old Australian has been stuck inside Ecuador's London embassy since June 2012 to avoid a British extradition to Sweden, which wants to question him on allegations of sexual assault.
The detention order was issued by prosecutors in 2010.
Assange denies the allegations and says he fears that if Britain extradited him to Sweden he would then be extradited to the United States where he could be tried for one of the largest information leaks in U.S. history.
The court said in a statement that the prosecutors' decision to question Assange in London supported the ruling to uphold the detention order.
"We are of course disappointed, and critical of the Supreme Court's way of handling the case.
This decision has been taken without letting us close our argument," Assange's lawyer Per Samuelson told Reuters.
Prosecutors first insisted Assange should come to Sweden for questioning, but in a U-turn in March agreed to conduct the interview in London.
One of the five Supreme Court judges dissented and argued for the arrest warrant to be lifted. Even if Sweden drops the investigation, Assange faces arrest by British police for jumping bail granted while the British courts considered the European arrest warrant issued by Sweden.
(Reporting by Daniel Dickson and Sven Nordenstam; Editing by Alistair Scrutton)
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