The decision came nearly two weeks after Mr Besigye lost presidential elections to President Yoweri Museveni.
High court Judge John Bosco Katutsi said prosecutors had not proved the defendant was guilty of the alleged 1997 assault.
"I find that the prosecution failed to prove their case. I hearby acquit the accused," he said in a ruling.
"The evidence before this court is inadequate and no court or judge with a sound mind can convict a person on such evidence," Judge Katutsi said.
Assessors who were monitoring the rape trial had earlier this month recommended that the presiding judge acquit Mr Besigye, who refused to put up a defence.
Under Ugandan law, assessors function like jurors but their advice is not binding on the judge, who may accept or disregard their findings.
Death sentence
Judge Katutsi said it appeared that police filed the charges before gathering enough evidence to sustain the rape charge of which, if convicted, Mr Besigye would have faced a maximum sentence of death.
The judge ruled that the complainant, Joanita Kyakuwa, who was also the prosecution's chief witness lied to the court.
Although he has been acquitted of the rape charges Mr Besigye along with 22 co-defendants now faces another trial on treason charges that is set to resume on March 15.
But Mr Besigye has consistently denied all the charges and maintains they were politically motivated attempts to either prevent him from running or hinder his ability to campaign for the February 23 elections.
The angry opposition Forum for Democratic Change (FDC) leader said the rape charge was as a result of "malicious prosecution" that had an adverse effect on his party and family.
He announced plans to plans ro sue to state for damages of which, if he get, would donate to women organisations in the east African nation.
Mr Museveni won re-election with nearly 60 percent of the vote in Uganda's first multi-party polls since 1980, extending his 20-year hold on power, while Mr Besigye took 37 percent of the vote.