In this study, we have investigated the contribution of bovine serum albumin (BSA) as the template for electrochemically synthesized poly(3,4-ethylenedioxythiophene) (PEDOT) film on a platinum (Pt) electrode. The electrode was capable to effectively adsorb the nano Au particles (AuNPs) to form a uniform layout, which was then able to immobilize the horseradish peroxidase (HRP) to construct a functional HRP/AuNPs/PEDOT(BSA)/Pt biosensor. The biosensor has displayed the nice linear correlations between the electrochemical current and the concentration of H2O2 with either cyclic voltammetry (CV) or differential pulse voltammetry (DPV). Meanwhile, we have provided the detail characterization of the HRP/AuNPs/PEDOT(BSA)/Pt biosensor. It is generally believed that AuNPs do not spontaneously attach to the PEDOT(PSS) film, however, our results have demonstrated that the AuNPs could easily and stably immobilized on the surface of PEDOT(BSA) film. Scanning electron microscopy (SEM) has revealed the uniform distribution of AuNPs on the PEDOT(BSA) film as well as the stability of the PEDOT film. Fourier transform infrared spectroscopy (FTIR) and Raman spectroscopies have not only confirmed the synthesis of conductive PEDOT where BSA servers as the template for the polymerization, but also clarified the interaction between AuNPs and PEDOT. X-ray photoelectron spectroscopy (XPS) has provided further insights into the stable immobilization of AuNP on the PEDOT(BSA) film. The current technology demonstrates a feasible procedure to form a functional and biocompatible AuNPs/PEDOT(BSA) film that has the potential applications in fabrication of various biosensors and electric devices. Keywords: bovine serum albumin; poly(3,4-ethylenedioxythiophene); gold nano particle; HRP biosensor;