We demonstrated a facile one-step synthesis strategy for the preparation of a large-scale reduced graphene oxide multilayered film doped with gold nanoparticles (RGO/AuNP film) and applied this film as functional nanomaterials for electrochemistry and Raman detection applications. The related applications of the fabricated RGO/AuNP film in electrochemical nonenzymatic H2O2 biosensor, electrochemical oxygen reduction reaction (ORR), and surface-enhanced Raman scattering (SERS) detection were investigated. Electrochemical data indicate that the H2O2 biosensor fabricated by RGO/AuNP film shows a wide linear range, low limitation of detection, high selectivity, and long-term stability. In addition, it was proved that the created RGO/AuNP film also exhibits excellent ORR electrochemical catalysis performance. The created RGO/AuNP film, when serving as SERS biodetection platform, presents outstanding performances in detecting 4-aminothiophenol with an enhancement factor of approximately 5.6 × 10(5) as well as 2-thiouracil sensing with a low concentration to 1 μM. It is expected that this facile strategy for fabricating large-scale graphene film doped with metallic nanoparticles will spark inspirations in preparing functional nanomaterials and further extend their applications in drug delivery, wastewater purification, and bioenergy.