Graphene oxide (GO) has drawn worldwide attention in various biomedical fields because of its unique properties, and great progress has been made in the past years. Probing the interaction between GO and proteins, understanding and evaluating potential impact of GO on the protein structure and function, is of significant importance for design and optimization of functional interfaces and revealing the bioeffect of GO materials. Cytochrome c (cyt c), one of the key components of respiratory chain, has played important roles in energy generation/consumption and many cellular processes including growth, proliferation, differentiation, and apoptosis. In this study, by combination of solution chemistry and spectroscopy, we systematically studied the interfacial interaction between GO and cyt c. Results suggest that GO could slightly perturb the active site of cyt c, enhancing its peroxidase activity. Structure of the active site is obviously changed with elapsed time, which in turn reduces peroxidase activity. Further study suggests that adsorption of cyt c on GO and the resulted structure change is a complex process resulting from the cooperation of various interaction forces. Hydrophobic interaction and π-π stacking, as well as electrostatic attraction, only slightly perturb the microenvironment of the active site of cyt c while hydrogen-bonding interaction is the main driving force for the structural change of the active site. Furthermore, long range electrostatic attraction between GO and cyt c may facilitate the short range hydrogen-bonding interaction, which intensifies the hydrogen-bonding-induced structural change. In addition, cyt c is partially reduced by GO in an alkaline environment. Based on the understanding of interfacial interaction mechanism between GO and cyt c, stable nanocomposites with enhanced peroxidase activity are successfully constructed by modulating the interfacial interaction forces. This work not only deepens the understanding of interaction between GO and functional protein, but also is of great importance for designing and applying of GO-based biomaterials.