Biocompatible polyetheretherketone (PEEK) is a favorable material for hard tissue repair due to its similar elastic modulus to that of the human bone. In this work, graphene oxide (GO) reinforced PEEK nanocomposites with different GO loading have been prepared by injection molding. Mechanical testing reveals that the toughness of the reinforced composite varies with the GO loading, with 0.5% GO giving the greatest elongation at break (86.32% greater than pristine PEEK). The underlying toughening mechanism has been attributed to the well-dispersed GO forming π-π* conjugations at the GO / PEEK interface. These conjugations also acted as the nucleation sites for oriented crystallized region in PEEK. As the GO content increases further (e.g > 0.5%), the fillers tend to agglomerate and would disturb the crystallites of PEEK and serve as stress concentration sites, resulting in decreased toughness. The biocompatibility of the composites has been evaluated in vitro, and the results showed that the addition of GO into PEEK favors the adhesion and spreading of bone marrow stromal stem cells, demonstrating the strong potential of our GO reinforced PEEK composites in applications such as hard tissue repair and replacement.