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The unvaccinated tennis star, who was on Thursday drawn in a first-round match for next week's Australian Open, landed a week ago only to be detained by border officials.
Immigration Minister Alex Hawke used his discretionary powers to cancel the visa after considering evidence provided by Djokovic's lawyers on the matter.
Australia's pandemic response has included an insistence that a visa holder must be double-vaccinated or show acceptable proof they cannot be vaccinated to enter quarantine-free.
On Thursday, Djokovic drew fellow Serb Miomir Kecmanovic for his first-round match.
Australian Border Force officials cancelled the world No.1's visa for entering the country while unvaccinated, only for the cancellation to be later quashed by a federal court.
Djokovic's lawyers, who are likely to challenge the latest cancellation in court, have provided lengthy submissions and supporting documentation to the minister.
Officials looked into potential discrepancies on Djokovic's declaration form, which stated he did not travel out of the country in the two weeks before his flight to Australia.
Djokovic was filmed playing tennis in Serbia on Christmas Day and was later seen training in Spain on December 31, both in the two-week window.
However, Djokovic has denied he was trying to mislead the government on the form, stating an agent had made an "administrative mistake" while filling out the form.
In a statement posted to social media, the Serbian player also admitted to attending a media interview in Belgrade when he knew he had COVID.
After carrying out a PCR test on December 16, Djokovic attended the interview two days later.
He said he had social distanced and wore a mask except when his photograph was being taken.
And while he went home after the interview to isolate for the required period, upon reflection this was an error of judgment, he said.
Since he was freed from immigration detention on Monday, Djokovic has been training at Melbourne Park.
The 34-year-old is looking to win a record 21st grand slam title when the Australian Open begins on Monday.
Labor leader Anthony Albanese said it was important that travellers to Australia followed the rules.
"They have never answered the question of how is it that that visa was granted in the first place if he wasn't eligible because he wasn't fully vaccinated."