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James Watson, 42, was jailed for a minimum term of 15 years last June after he was found guilty of strangling the schoolboy to death in woods near his home in Peterborough, following a trial at the Old Bailey.
Watson, who was aged 13 at the time of the murder, challenged his conviction at the Court of Appeal, claiming there was a "wholesale loss and destruction" of evidence.
His lawyers also complained about the trial judge allowing so-called "bad character" evidence, which suggested Watson had a sexual interest in young boys and in strangulation, to be considered by the jury.
But he lost the appeal in a written ruling delivered by three judges on Monday.
Lord Justice Holroyde said: "We are satisfied that the judge did not err in admitting the bad character evidence.
"The weight to be given to the evidence was then a matter for the jury."
Read more:
Mum's rage as man who murdered her son, six, almost three decades ago is jailed
James Watson found guilty of murdering Rikki Neave nearly 30 years ago
The murder was among the most high-profile cold cases in Britain until DNA was identified on the victim's clothes following a re-examination of the evidence two decades later.
Watson's trial heard how he strangled Rikki with his own jacket before stripping the boy's body and posing him in a "star shape".
Prosecutors said a "key piece" of evidence was the DNA he left on Rikki's clothes. Samples were taken in 1994 but technology was not sufficiently advanced to provide a match until 2015.
Watson, who denied murder, told police he lifted Rikki up so the youngster could see over a fence.
Sentencing him last year, the trial judge, Mrs Justice McGowan said the law meant Watson had to be handed a minimum term relevant to his age at the time of the killing.
Rikki's mother, Ruth Neave, said in a statement: "The news today brings to an end a very long journey of horror and tragedy."
She was found not guilty of the boy's murder following a trial at Northampton Crown Court in 1996 - although she was given a seven-year jail term after admitting child cruelty.