The American briefly addressed the appeals court at the end of a hearing in Perugia, central Italy.
Knox was found guilty in 2009 of sexually assaulting and murdering Meredith Kercher, 21, and was sentenced to 26 years in prison.
"I've spent more than three and a half years in prison as an innocent person, and this for me is very frustrating and mentally exhausting," Knox said. "But nothing is more important than finding the truth after prejudices and many mistakes."
"I don't want to spend my whole life in prison as an innocent," the 23-year-old added.
Knox and her co-defendant, Italian Raffaele Sollecito, were arrested on 6 November 2007, a few days after Kercher's body, her throat slit, was found in the apartment she and Knox shared as exchange students in Perugia.
Sollecito, who was Knox's boyfriend at the time of the murder, was convicted of the same charges and sentenced to 25 years' jail.
The two have always denied wrongdoing. Their appeals trial is ongoing and a lot hinges on an independent review of DNA evidence used to convict them.
Yesterday the court gave independent experts carrying out the review until 30 June to finish - over a month after the original 21 May deadline.
Ivorian-born Rudy Guede, 23, is serving 16 years in an Italian jail for sexually assaulting and murdering Kercher.