An electrochemical hydrogen peroxide biosensor was designed by immobilizing horseradish peroxidase (HRP) on Ag nanoparticles/cysteamine/p-aminobenzene sulfonic acid/glassy carbon (GC) electrode. Ag nanoparticles can act as tiny conduction centers on electrodes that adsorb redox enzymes, facilitating the transfer of electrons with no requiring any loss of biological activity. The forerunner film was first electropolymerized on the glassy carbon electrode with p-aminobenzene sulfonic acid (p-ABSA) by cyclic voltammetry. The cysteamine (CA) was bound on the surface of the film by electrostatic force, then Ag nanoparticles were immobilized on the cysteamine monolayer, and lastly HRP was adsorbed onto the surfaces of the Ag nanoparticles. A dramatic decrease in the overvoltage of H2O2 was observed with improved sensitivity, which makes the modified electrodes of great promise for oxidase-based amperometric biosensors. The biosensor responded to H2O2 in the linear range from 1.2×106 mol/L to 9.8×103 mol/L with a detection limit of 1.1×108 mol/L. Moreover, the obtained biosensor exhibited good accuracy and high sensitivity.