Background: Graphene oxide (GO)can be dispersed through functionalization, or chemically converted to make different graphene-based nanocomposites with excellent mechanical and thermal properties. Chitosan, a partially deacetylated derivative of chitin, is extensively used for food packaging, biosensors, water treatment, and drug delivery. GO can be evenly dispersed in chitosan matrix through the formation of amide linkages between them, which is different from previous reports focusing on preparing GO/chitosan nanocomposites through physical mixing. Results: In this study, free-standing graphene oxide-chitosan (GO-chitosan) nanocomposite films have been prepared. The GO-chitosan films are biologically compatible and mechanically reinforced. Through the formation of amide linkages between GO’s carboxylic acid groups and chitosan's amine groups, GO could be evenly dispersed within the chitosan matrix. We also characterized the GO-chitosan composite films using element analysis, Fourier transform infrared spectroscopy, X-ray photo electron spectroscopy, differential scanning calorimetry, and thermo gravimetric analysis. Compared to pristine chitosan film, the tensile strength of GO-chitosan film is improved by 2.5 folds and Young’s modulus increases by nearly 4.6 folds. The glass transition temperature of GO-chitosan composite film shifts from 118°C to 158°C compared to the pristine chitosan, indicating its enhanced thermal stability. GO-chitosan composite film was also evaluated for its biocompatibility with C3H10T1/2 cells by in vitro fluorescent staining. The graphene oxide-reinforced chitosan composite films could have applications in functional biomaterials. Conclusion: The present study describes a useful and simple method to chemically attach biocompatible chitosan onto graphene oxide. We envision that the GO-chitosan film will open avenues for next-generation graphene applications in the realm of functional biomaterial.