An amperometric hydrogen peroxide (H 2 O 2 ) biosensor was developed based on the immobilization of horseradish peroxidase (HRP) onto gold nanoparticles (GNPs)-adsorbed conducting poly(brilliant cresyl blue) (PBCB) film. The modification process was characterized by field emission scanning electron microscope (FE-SEM) and electrochemical impedance spectroscopy (EIS). The effects of experimental parameters such as the concentration of the mediator (hydroquinone, HQ), pH of the solution, and the working potential were investigated for optimum analytical performance. In the presence of the mediator, the immobilized HRP showed an excellent electrocatalytic activity towards the reduction of H 2 O 2 . The linear dynamic range from 5 to 150 μM with the regression coefficient of 0.99 was obtained. The detection limit was calculated to be 0.5 μM based on a signal-to-noise ratio of 3. The reproducibility and stability of the biosensor were studied, with satisfactory results. The biosensor performance was evaluated with respect to possible interferences and the application to real sample analysis.