In this study, we describe the use of the combination of eletrografting poly(N-mercaptoethyl acrylamide) and Au nanoparticles in the construction of high-performance biosensors. The poly(N-mercaptoethyl acrylamide) was electrografted onto the glassy carbon electrode surface, which provided a strongly adhering primer film for the stable attachment of Au nanoparticles and horseradish peroxidase (HRP) enzymes. The performances of the biosensors based on the HRP immobilized in the Au/poly(N-mercaptoethyl acrylamide) composite film were investigated. A couple of redox peaks were obtained, indicating that the Au nanoparticles could facilitate the direct-electron transfer between HRP and the underlying electrode. The biosensor showed an excellent electrocatalytic activity toward the reduction of hydrogen oxide and long-term stability, owing to the stable electrografted film and biocompatible Au nanoparticles. Our results demonstrate that the combination of electrografting and Au nanoparticles provides a promising platform for the immobilization of biomolecules and analysis of redox enzymes for their sensing applications.