This work describes a new one-step large-scale electrochemical synthesis of graphene/polyaniline (PANI) composite films using graphite oxide (GO) and aniline as the starting materials. The size of the film could be controlled by the area of indium tin oxide (ITO). Scanning electron microscopy (SEM), transmission electron microscopy (TEM), X-ray photoelectron spectroscopy (XPS), Fourier transform infrared spectroscopy (FTIR), and ultraviolet–visible absorption spectrum (UV–vis) results demonstrated that the graphene/PANI composite film was successfully synthesized. The obtained graphene/PANI composite film showed large specific area, high conductivity, good biocompatibility, and fast redox properties and had perfect layered and encapsulated structures. Electrochemical experiments indicated that the composite film had high performances and could be widely used in applied electrochemical fields. As a model, horseradish peroxidase (HRP) was entrapped onto the film-modiﬁed glassy carbon electrode (GCE) and used to construct a biosensor. The immobilized HRP showed a pair of well-deﬁned redox peaks and high catalytic activity for the reduction of H2O2. Furthermore, the graphene/PANI composite film could be directly used as the supercapacitor electrode. The supercapacitor showed a high specific capacitance of 640 F g−1 with a retention life of 90% after 1000 charge/discharge cycles.