Knox was convicted in 2009 of sexually assaulting and murdering her British roommate, Meredith Kercher in 2007.
She was sentenced to 26 years in prison but denies any wrongdoing.
Also on trial is Knox's Italian former boyfriend Raffaele Sollecito, who was sentenced to 25 years behind bars.
Both have appealed the guilty verdict.
The appeals trial formally opened last month but that hearing was immediately adjourned. With Saturday's hearing, the new proceedings got into full swing.
"I am innocent. Raffaele is innocent. We did not kill Meredith," Knox said during her 20-minute address, speaking Italian, and her voice breaking. "It doesn't' do justice to Meredith and her loved ones to take our lives from us."
Knox had addressed the court in the previous trial but never for as long or as passionately. She said she regretted not being able to fully speak her mind before, saying that words don't come easily to her and that she has a difficult time standing up for herself.
The Associated Press contributed to this report.