The decision likely means that Djokovic, who is not vaccinated against Covid-19, will remain in detention in Melbourne until he is deported.
Tennis world number one Novak Djokovic has said in a statement that he was "extremely disappointed" with the decision to cancel his visa to enter Australia, but would cooperate with the authorities in relation to his departure from the country.
The Serbian champion on Sunday said he hoped the focus would now return to tennis and the Australian Open Grand Slam, which starts in Melbourne on Monday.
Djokovic had appealed Immigration Minister Alex Hawke's decision to cancel his visa on the grounds that he was a threat to public order because his presence would encourage anti-vaccination sentiment amid Australia's worst Covid-19 outbreak.
"I will now be taking some time to rest and to recuperate, before making any further comments beyond this," the 34-year-old said in a statement after the court dismissed his case.
"I am extremely disappointed with the Court ruling to dismiss my application for judicial review of the Minister's decision to cancel my visa, which means I cannot stay in Australia and participate in the Australian Open.
"I respect the Court's ruling and I will cooperate with the relevant authorities in relation to my departure from the country."
Djokovic, who is not vaccinated against Covid-19, was granted a medical exemption to compete at the Melbourne Park major, a tournament he has won nine times including the last three editions.
His fate was finally decided after a rollercoaster 10 days that saw him detained by authorities, released and then detained again ahead of the Australian Open, which starts on Monday.
Rafa Nadal, who is tied with Djokovic on 20 major titles, said "too many questions" still needed to be answered concerni ng the Serb's bid to play the tournament, while other leading players lamented how Slam has been overshadowed by the saga.
"I am uncomfortable that the focus of the past weeks has been on me and I hope that we can all now focus on the game and tournament I love," Djokovic said, while also thanking those that supported him.
"I would like to wish the players, tournament officials, staff, volunteers and fans all the best for the tournament."
A deportation order on Sunday usually also includes a three-year ban on returning to Australia.
The minister cancelled the visa on the grounds that Djokovic’s presence in Australia may be a risk to the health and “good order” of the Australian public and “may be counterproductive to efforts at vaccination by others in Australia.”
Djokovic’s visa was initially cancelled on January 6 at Melbourne’s airport hours after he arrived to compete in the first Grand Slam of 2022.
A border official cancelled his visa after deciding Djokovic didn’t qualify for a medical exemption from Australia’s rules for unvaccinated visitors.