An amperometric hydrogen peroxide (H2O2) biosensor was developed based on the immobilization of horseradish peroxidase (HRP) onto a gold-nanoparticle (GNP)-adsorbed conducting poly(thionine) (PTH) film. The modification process was characterized by scanning electron microscope (SEM), electrochemical impedance spectroscopy (EIS) and cyclic voltammetry (CV). The effects of experimental parameters such as the concentration of the mediator (hydroquinone, HQ), the pH of the solution, and the working potential were investigated for optimum analytical performance. In the presence of the mediator, the immobilized HRP showed excellent electrocatalytic activity towards the reduction of H2O2. The linear dynamic range of 5-150 microM with the regression coefficient of 0.999 was obtained. The detection limit was calculated to be 1.5 microM based on a signal-to-noise ratio of 3. The reproducibility, repeatability, and stability of the biosensor were studied, with satisfactory results. The biosensor performance was evaluated with respect to the possible interferences and the application of the biosensor to real-sample analysis.