Graphene was prepared successfully by introducing -SO3- to separate the individual sheets. TEM, EDS and Raman spectroscopy were utilized to characterize the morphology and composition of graphene oxide and graphene. To construct the H2O2 biosensor, graphene and horseradish peroxidase (HRP) were co-immobilized into biocompatible polymer chitosan (CS), then a glassy carbon electrode (GCE) was modified by the biocomposite, followed by electrocleposition of Au nanoparticles on the surface to fabricate Au/graphene/HRP/CS/GCE. Cyclic voltammetry demonstrated that the direct electron transfer of HRP was realized, and the biosensor had an excellent performance in terms of electrocatalytic reduction towards H2O2. The biosensor showed high sensitivity and fast response upon the addition of H2O2, under the conditions of pH 6.5, potential -0.3 V. The time to reach the stable-state current was less than 3 s, and the linear range to H2O2 was from 5 x 10(-6) M to 5.13 x 10(-3) M with a detection limit of 1.7 x 10(-6) M (S/N = 3). Moreover, the biosensor exhibited good reproducibility and long-term stability. (C) 2010 Elsevier Ltd. All rights reserved.