Direct electron transfer (DET) in biocatalysts and the interactions of biocatalysts at electrode interfaces are critical issues for the development of electrochemical devices. In comparison to high-performance complex electrodes, graphene-based electrodes have attracted significant attention based on their superior electrical conductivity, material properties, and low cost. However, the immobilization of laccase (LAC), an oxygen-reducing enzyme with high catalytic activity that is applied to cathodes, and interfaces formed between LAC and graphene have rarely been explored. In this study, electrochemical experiments employing cyclic voltammetry and electrochemical impedance spectroscopy were performed, and it was determined that graphene exhibits a maximum of a 1.57-fold increase in terms of its oxygen reduction rate compared to Au and carbon nanotubes. Additionally, DET rate revealed that graphene behaves more efficiently on immobilized LAC. Furthermore, absorbed morphologies were visualized, and computational methods were applied to verify binding sites, orientations, structures, and binding affinities in atomic scale. The axial ligands at T1 Cu sites were mutated using different hydrophobic amino acids, and the effects of mutation on interactions at interfaces were compared. Based on our experimental and theoretical results, LAC immobilization on graphene appears to be stronger than that on a charged surface without critical structural changes.