Andrew John Cobby was found guilty in a 2021 trial of beating and strangling his estranged wife Kym, 51, outside her Gold Coast hinterland home in November 2017.
Justice Thomas Bradley on Thursday handed down the appeal judgment in which he and two other justices found "this was not a case where there was a significant possibility that an innocent man was convicted".
Cobby represented himself before the Brisbane Court of Appeal and in June 2024 spent several hours presenting what he said were six grounds that his guilty verdict should not stand and he should get a retrial.
Cobby claimed retested DNA evidence on the hammer would have been of "extreme importance" to his trial's jury.
"What would the jury make of the evidence before us today? Only the jury can make that determination," he said.
He said DNA retesting of the hammer that was presented at trial as the murder weapon had cast doubt over its link to a man he was living with at the time of Ms Cobby's death.
The appeal justices were also shown pictures of the hammer which Cobby claimed showed his housemate had incorrectly described its colour and shape.
Appeal justices Bradley, Peter Flanagan and Debra Mullins on Thursday rejected all six of Cobby's grounds for appeal and ruled he had failed to show the verdict of guilty was unreasonable.
"(Cobby) has failed to demonstrate that the fresh evidence of the results of the further DNA testing since the appellant's trial gave rise to a significant possibility that a jury, acting reasonably, would have acquitted (Cobby) if the fresh evidence had been adduced at the trial," Justice Mullins stated.
Cobby, then aged 59, was sentenced to life imprisonment for what trial Justice Peter Callaghan said was a clearly premeditated and sickening crime.
He admitted being present during the attack on the woman he had married more than three decades earlier - although they mostly lived apart from 2003 - but denied being responsible.
Instead, Cobby told police an unknown assailant was behind the killing, ambushing his wife as he was about to get into a red Chrysler he had borrowed.
Philip McCarthy KC for the prosecution told the appeal justices in 2024 that the new DNA results for the hammer were not materially different from what was presented at trial.
"The hammer is one strand amid 13 strands of evidence, one of which being that Cobby's clothing was saturated with the blood of his wife," Mr McCarthy said.
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