Rachel Fee, 32, argued the judge in her 2016 trial misdirected the jury about the lesser charge of culpable homicide before they considered their verdict.
Two-year-old Liam Fee died at his home near Glenrothes on 22 March 2014.
Fee, also known as Trelfa, and her civil partner Nyomi Fee, 30, were both convicted of abusing and murdering the toddler.
He had suffered fatal heart injuries similar to those found on road crash victims and spent the last few days of his short life in agony from an untreated broken leg and fractured arm.
The pair - originally from Ryton, Tyne and Wear - were also convicted of being behind a catalogue of cruelty against two boys in their care, one of whom they tried to blame for Liam's death.
Judge Lord Burns last year handed both women life sentences and ordered Rachel Fee to serve a minimum of 23-and-a-half years behind bars while "domineering" Nyomi Fee was sentenced to spend at least 24 years in prison.
Senior judges at the Court of Criminal Appeal in Edinburgh heard arguments from Fee's legal team and the Crown last month, and have now rejected the appeal in a written opinion.
Fee's appeal centred on what her defence team said was a misdirection by the trial judge during his legal summing-up at the High Court in Livingston in May.
Defence QC Brian McConnachie told the court the judge did not present to the jury with "the possibility that they could convict Nyomi Fee of murder and convict the appellant (Rachel Fee) of culpable homicide".
The lawyer submitted there was a case, on the evidence, for the jury to identify co-accused Nyomi Fee as having been the main "actor" in committing the murder.
Advocate depute Alex Prentice QC, for the Crown, argued it was a "clear case in which the Crown had established concert for murder".
The appeal verdict, delivered by Lord Turnbull, concluded: "There is therefore no basis upon which it could be said that the appellant did anything less than actively associate herself with a common criminal purpose which included the taking of human life, or carried the obvious risk that human life would be taken, in the carrying-out of which murder was committed.
"In the circumstances of this case the evidence led by the Crown clearly established that the appellant had associated herself with a purpose which carried with it the obvious risk that life would be taken.
"We therefore agree with the trial judge and the advocate depute that the directions argued for by the appellant were correctly omitted.
"For the reasons which we have given, the appeal against conviction is refused."

Residents will be moved from their homes in the south-western province of Guizhou to prevent interference with the telescope's electromagnetism.
The project's lead scientists told China's state news agency that the telescope would further the search for intelligent life in the universe.
It will dwarf the world's current largest such telescope in Puerto Rico.
The 500m-wide Aperture Spherical Radio Telescope (FAST) is due to be operational this year.
Provincial officials have vowed to relocate 9,110 residents living within five kilometres of the listening device by September, the official Xinhua news agency said.
The relocations will "create a sound electromagnetic wave environment", the news agency cited regional official Li Yuecheng as saying.
Residents will reportedly receive 12,000 yuan (Â£1,280; $1,800) in subsidies for their troubles, with some getting extra support for housing, it said.
FAST, built at a cost of 1.2bn yuan, will dwarf the Arecibo Observatory in Puerto Rico as the world's largest radio telescope, which is about 300m in diameter.
Wu Xiangping, director-general of the Chinese Astronomical Society, said the telescope's high level of sensitivity "will help us to search for intelligent life outside of the galaxy", Xinhua reported.
In the past China has relocated hundreds of thousands of people to make way for large infrastructure projects such as dams and canals. Many have complained of poor compensation.
As well as upping investment in astronomy, Beijing is accelerating its multi-billion-dollar space exploration programme, with plans for a permanent orbiting station by 2020.
A mother jailed for life for murdering her toddler son in Fife has had her appeal against conviction rejected.
[NEXT_CONCEPT]
China is preparing to relocate nearly 10,000 people to make way for the world's largest radio telescope.