A novel hydrogen peroxide biosensor based on horseradish peroxidase/GNPs-thionine/chitosan has been developed. Gold nanoparticles fixed with horseradish peroxidase were adsorbed on glassy carbon electrode by the chitosan which cross-linked with the electron mediator of horseradish peroxidase as the bridge linking agent. The assembly procedures were monitored by UV-visible spectral scanning, biolayer interferometry, cyclic voltammetric and alternating current impedance. The chronoamperometry was used to measure hydrogen peroxide. The hydrogen peroxide biosensor linear range of detection is 1 x 10(-7)-1 x 10(-4) mol/L, detection limit up to 5.0 x 10(-8) mol/L. Moreover the stability, reproducibility and selectivity of the biosensor were also studied and the results confirmed that the biosensor exhibit fast response to hydrogen peroxide and possess high sensitivity, good reproducibility and long-term stability. (C) 2011 Elsevier Ltd. All rights reserved.