With just a day to go before the first tennis Grand Slam of the year gets underway, Djokovic has been ordered to leave the country and will be unable to defend his men's singles title.

Djokovic won an appeal against his deportation last week after he was forced to spend several days in an immigration detention centre upon arrival in Australia.

However, the 34-year-old - who spent last night in the detention hotel - failed to overturn Australian Immigration Minister Alex Hawke's decision to cancel his visa again, citing "health and good order grounds".

The Australian Government argued that the presence of Djokovic, who is not vaccinated against COVID-19, in the country may lead to "an increase in anti-vaccination sentiment".

The visa cancellation unanimously upheld by the Federal Court of Australia, although the reasons behind the decision made by the three judges are yet to be released.

Djokovic, a nine-time Australian Open champion, has been ordered to pay costs and could be denied entry into Australia for three years.

The cancellation of an Australian visa results in a three-year ban from the country unless there are "compelling circumstances" which impact the interests of Australia.

Djokovic was due to face fellow Serbian Miomir Kecmanović in the opening round of the men’s singles tomorrow, but he is now due to be replaced by a lucky loser Salvatore Caruso from Italy.

Speaking after the verdict, Djokovic said he was "extremely disappointed" with the court's ruling and planned to take "some time to rest and recuperate" before making further comment. 

"I respect the court's ruling and I will cooperate with the relevant authorities in relation to my departure from the country," the statement from Djokovic read.

"I am uncomfortable that the focus of the past weeks has been on me and I hope we can all now focus on the game and tournament I love."

The 20-time Grand Slam champion initially landed in Australia on January 5, having obtained an exemption from tournament organisers to play in the Australian Open while being unvaccinated.

It is claimed that Djokovic had COVID-19 on December 16, which medical panels assembled by Tennis Australia and the Victorian Government accepted was grounds for an exemption.

However, border officials ruled that Djokovic had failed to meet vaccination requirements to enter Australia - a separate process - and designated him for deportation.

After staying in a detention centre over that weekend, Djokovic's legal team successfully appealed that decision.

A Federal Circuit and Family Court judge ruled that Djokovic had not been given enough time at the airport to speak with his legal team in response to the move to revoke his visa.

Djokovic took to the practice court at Melbourne Park in preparation for the Australian Open only for his visa to be cancelled again after Hawke exercised his personal power to end the tennis star's stay in the country.

Australian Government lawyers had argued that Djokovic could pose a health risk to Australians by causing "an increase in anti-vaccination sentiment".

Nick Wood, representing Djokovic, told the court today that there was "not a single line of evidence in the material provided any specific or logical foundation whatsoever that the mere presence of Mr Djokovic in Australia in itself may somehow foster anti-vaccination sentiment".

"The clearest and most attractive window through which to view the error is the public-interest lens," added Wood.

"It is irrational or unreasonable to look at only one side of the coin."

However, Stephen Lloyd, representing Hawke, claimed Djokovic’s presence in Australia would "encourage people to emulate his position" on COVID-19 vaccines.

"His ongoing non-vaccination status is open to infer that a person in the applicant's position could have been vaccinated if he wanted to be," said Lloyd.

"Even before vaccines were available he was against it - his prima facie position was to be against them.

"He's a high-profile person who is in many respects a role model for many people.

"His presence in Australia would present more strongly to Australians his anti-vaccination views."

The controversy surrounding Djokovic’s participation at the Australian Open has created a diplomatic row, with Serbian President Aleksandar Vučić hitting out at Australia for the "harassment of the world’s best tennis player".

On top of the legal disputes in Australia, Djokovic is being investigated for breaking COVID-19 rules in both Serbia and Spain.

This includes attending an in-person interview and photoshoot with French newspaper L'Équipe when he had tested positive for coronavirus .

The tournament begins tomorrow, with crowds at the show courts set to be restricted to 50 per cent capacity due to COVID-19 restrictions.