After a brief hearing, Florence's appeals court ruled inadmissible a request from Ivorian Rudy Guede for an extraordinary review of his case in light of the acquittal of Knox and her former boyfriend, local media reported.
Lawyers for Guede, who was convicted after a fast-track trial in 2008, had presented the court with a deposition arguing that the terms of the 2015 acquittal of Knox and her former boyfriend Raffaele Sollecito made their client's conviction unsafe.
But the judge in the case ruled that their was no basis for the case to be reopened and ordered Guede to pay the costs of the hearing.
The ruling means Guede's 16-year sentence for Kercher's murder, imposed after he appealed an initial 30-year jail term, stands, at least for the time being.
A lawyer for Guede was quoted as telling reporters he would consider appealing Tuesday's ruling in Italy's highest appeal court, the Court of Cassation.
The ruling was the latest twist in the lengthy legal saga that began with 21-year-old Kercher's half-naked body being discovered in a pool of blood in a back room of the house she shared with Knox in Perugia, central Italy.
The student's throat had been slashed and she had been stabbed 47 times.
A year after the judge in Guede's case said he could not have acted alone, Knox and Sollecito were also convicted of the murder.
But that verdict was overturned on appeal in 2011 and Knox and Sollecito were released after four years in prison.
Another court then ordered a retrial which reinstated the original convictions only for the Court of Cassation to throw out all charges against the pair in March 2015.
Six months later the court released a written judgement which said the ruling reflected "major flaws" in the police's handling of the investigation, the absence of a "body of evidence" allowing for a safe conviction and the absence of any admissible DNA evidence linking the two to the grisly murder.
Legal experts said at the time that the Knox-Sollecito acquittal was tantamount to saying Guede acted alone, in contradiction of the judge's summing up in his first trial.
Tommaso Pietrocarlo, a member of Guede's defence team, told reporters the Florence court had said there was no contradiction between the two judgements, despite this being "rather obvious," in his view.
In particular, Pietrocarlo said Guede's conviction partly rested on evidence that he had used Sollecito's knife to kill Kercher while the judge who acquitted Knox and her former boyfriend had ruled that it was not the murder weapon.