The 68-year-old was sentenced to 23 years in jail for killing partner Bob Chappell aboard the couple's yacht, the Four Winds, on Australia Day 2009.
Her non-parole period expires on August 20, but she has previously indicated she'll remain in prison while her conviction stands.
Neill-Fraser's appeal before Tasmania's Court of Criminal Appeal was dismissed in November last year, with two of the three judges finding new evidence did not meet the required threshold of "fresh and compelling".
She applied to the High Court seeking special leave to appeal the judgment, but Justices Stephen Gageler, Simon Steward and Jacqueline Gleeson refused that on Friday.
The court stated that the principles applying to a second appeal based on fresh evidence are established.
But it was not persuaded there were sufficient prospects of demonstrating the majority of the Court of Criminal Appeal erred in applying those principles.
Neill-Fraser's appeal was centred around the whereabouts of then homeless teenager Meaghan Vass, whose DNA was found aboard the yacht.
It was put by the prosecution at the 2010 trial that the DNA deposit was a "red herring" and arrived on the boat via secondary transfer.
Ms Vass has given conflicting accounts about whether she was on the boat the night Mr Chappell was murdered.
She signed an affidavit saying she was on the yacht but later recanted, telling an appeal hearing she was coerced into giving that evidence.
Neill-Fraser's legal team abandoned the evidence of Ms Vass, which they had held up as the pillar of their case, during the appeal process.
Justice Helen Wood determined evidence of forensic expert Maxwell Jones, which Neill-Fraser's legal team claimed cast doubt on the secondary transfer scenario, did not prove there was a miscarriage of justice.
She said Mr Jones' evidence conformed significantly with what the jury heard at the trial.
In his dissenting view, Justice Stephen Estcourt determined there was a "significant possibility" the trial jury might have acquitted Neill-Fraser had Mr Jones' evidence been before it.
She previously had a special leave application refused by the High Court in 2012.
Neill-Fraser was found to have attacked Mr Chappell and dumped his body in Hobart's River Derwent but has maintained her innocence. Mr Chappell's body has never been found.
Her supporters said the High Court decision was another setback, but it had strengthened their resolve to "keep fighting for justice". They renewed calls for an independent judicial inquiry into the case.
Supporters are planning to rally outside the Tasmanian parliament on August 20, the day she was charged and incarcerated 13 years ago.
But Neill-Fraser Support Group president Rosie Crumpton-Crook told AAP on Friday they're not sure whether she'll leave prison on that day.
"That is a very personal decision for Sue, her family and legal team. We'll support her whatever decision she makes," Ms Crumpton-Crook said.
"She's worried the authorities will forget about her (if she leaves prison), but we have assured her we won't let that happen."
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