The now 24-year-old Palfreeman was found guilty two years ago of the December 2007 murder of Bulgarian law student Andrei Monov, 20, in the country's capital, Sofia.
A subsequent 20-year prison sentence was appealed by Palfreeman, who sought a retrial, maintaining he acted in self defence when he intervened to help two Roma gypsies being beaten by a group of boys, including Mr Monov.
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Prosecutors also sought a review, citing a violent knife crime and wanting the penalty increased to a life sentence without parole.
With the Sofia Court of Appeals failing to alter the sentence, the matter reached Sofia's Supreme Court of Cassation, which on Wednesday also confirmed the 20-year term.
The freejock.com website, jointly run by Palfreeman's father, Newcastle pathologist Dr Simon Palfreeman, had not been updated several hours after the release of Wednesday's decision.
The family of Palfreeman had previously made clear that they would approach the European Court of Justice if the court rejected their appeal.
The Australian profile of the case was boosted in June when twin episodes of ABC Television's Australian Story focused on Dr Palfreeman's struggle to help his son through a "corrupt" and "malfunctioning" Bulgarian judicial system.
"I did not attack 10 to 15 people for no reason," Palfreeman is quoted as saying during his time in court.
Sofia news agency noinvite.com which has closely monitored Palfreeman's court matters, reported that legal proceeding show "that the Bulgarian's death has resulted from a stab in the back, and has not been caused in self-defence".
The Palfreeman's have maintained that there were flaws with how evidence was presented to and interpreted by the Bulgarian courts.
A second man, Antoan Zahariev, who was wounded by Palfreeman during the same incident on December 28, 2007, told noinvite.com earlier this year that the Australian "was just trying to kill us. He was ready to attack anyone, who was closest to him".
In addition to his jail sentence, Palfreeman is ordered to pay 450,000 leva (approx A$299,890) )in compensation to the victims' families.
