The studio, which is hoping to reclaim the full rights to Superman, vowed to take the case to trial in a district court if their appeal is rejected.
Siegel's heirs hold a share of the license following a court victory in 2008. A judge ruled that Action Comics #1, which features the superhero's debut, was not produced as a "work for hire" for DC Comics.
DC and Warner Bros control all elements of Superman added after Action Comics #1, leaving the Siegel estate with the rights to the character's appearance, origin, and Clark Kent alter ego. The Shusters will have the opportunity to seize a share of the licence next year.
Warner's appeal reiterates all of its previous arguments in the long-running legal battle, stating that the company had a verbal agreement with the creators' heirs, violated by interference from lawyer Marc Toberoff. The firm also plans to present evidence that Action Comics #1 was a "work for hire".
The studio sued Toberoff based on evidence contained in sensitive documents that were stolen from his office and delivered to Warner anonymously. The material was later passed as admissible in court by a district judge.
A date for Warner Bros' appeal hearing is yet to be confirmed.