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World number one Djokovic, who is level with Roger Federer and Rafa Nadal on 20 Grand Slam titles, has declined to reveal his vaccination status, and said he is unsure if he will defend his Australian Open crown.
Clarifying Australia's visa requirements, Minister for Immigration Alex Hawke said foreign players would need to have had two vaccination shots to play the Grand Slam at Melbourne Park in January.
"You’ll need to be double vaccinated to visit Australia. That’s a universal application, not just to tennis players. I mean that every visitor to Australia will need to be double vaccinated,” Hawke told Australian Broadcasting Corporation radio.
"I don’t have a message to Novak. I have a message to everybody that wishes to visit Australia. He’ll need to be double vaccinated."
Apart from Serbian Djokovic, who has won nine of his Grand Slam titles at the Australian Open and the last three in succession at Melbourne Park, the rule could exclude scores of players from the tournament.
More than a third of professional players remain unvaccinated, according to recent media reports.
Both the men's ATP and women's WTA tours have urged players to get vaccinated but some have voiced reservations.
Russian men's U.S. Open champion Daniil Medvedev and German world number four Alex Zverev have expressed scepticism, although their vaccination status remains unknown.
Greek world number three Stefanos Tsitsipas said in August he would only get vaccinated if it became mandatory, though later said he planned to have shots by the end of the year.
Tennis Australia, which organises the Grand Slam, said it was working with authorities on conditions for players, fans and tournament staff.
"Our understanding is that the details around international visitors entering the country are yet to be decided and we hope to have more information soon," the governing body said.
Australia's health minister Greg Hunt said the country's rules were about protecting Australians.
"They apply to everyone without fear or favour. It doesn't matter whether you are number one in the world or you are anything else," he told a media conference on Wednesday.
Australia has shut its international borders to non-citizens and non-permanent residents for 18 months, though there have been some high-profile exceptions.
International travel is expected to begin for Australian citizens within weeks, but non-citizens are expected to be shut out until early-2022.
Authorities in Victoria state, which hosts the Australian Open, said they would not make special deals with unvaccinated athletes to allow them to compete even if they secured visas.
Melbourne, Australia's second largest city, has been locked down since August due to an outbreak of the highly infectious Delta variant but will begin to open up on Friday, when 70% of the adult population in Victoria is expected to be fully vaccinated.