Graphene was synthesized by a chemical method to reduce graphite oxide and well characterized by scanning electron microscopy (SEM), transmission electron microscopy (TEM), powder X-ray diffraction (PXRD) and Fourier transform infrared (FTIR) spectra. Horseradish peroxidase (HRP) immobilized on a graphene film glassy carbon electrode was found to undergo direct electron transfer and exhibited a fast electron transfer rate constant of 4.63 s(-1). The HRP-immobilized electrode was investigated by electrochemical impedance spectroscopy (EIS) and cyclic voltammetry (CV). The CV results showed that the modified electrode gave rise to well-defined peaks in phosphate buffer, corresponding to the electrochemical redox reaction between HRP-Fe(III) and HRP-Fe(II). The obtained electrode also displayed an electrocatalytic reduction behavior towards H2O2. The new H2O2 sensor shows a linear range of 0.33-14.0 mu M (R-2 = 0.9987) with a calculated detection limit of 0.11 mu M (SIN = 3). Furthermore, the biosensor exhibits both good operational storage and storage stability. (C) 2010 Elsevier Ltd. All rights reserved.