A simple and sensitive amperometric sensor was developed based on electrochemical co-reduction of graphene oxide/nanoparticle (ERGO-NP) composite films by chronoamperometrymethod on indium tin oxide (ITO) electrodes. By analyzing the properties of the composite films with experimental results and theoretical investigations, a comprehensive report on the physical and electrochemical interfacial properties of graphene mixed gold nanoparticles composite electrode is presented. The developed electrodes were applied for the detection of hydrogen peroxide (H2O2) based on the direct electrochemistry of horseradish peroxidase (HRP). Standard carbodiimide chemistry enabled the covalent tethering of HRP on to the modified film thus maintaining its native structures, consequently facilitating the direct electron transfer between the protein and the underlying surface. The electrochemical result revealed that the ERGO-NP/ITO electrodes exhibited much higher conductivity (ca. 5 times) when compared with unmodified electrode. The developed sensor using amperometric measurements revealed high sensitivity (1808.9 mu A.mM(-1) cm(-2)), with detection limit (0.6 mu M) achieved under optimized conditions toward H2O2. Further, the sensor showed good specificity, stability and reproducibility toward H2O2 detection, therefore results proved that well-dispersed, high surface area and highly conductive modified surface could be a promising material for protein adsorption and fabrication of electrochemical biosensors. (C) 2014 The Electrochemical Society. All rights reserved.