A disposable biosensor for determination of hydrogen peroxide (H2O2) based on Fe3O4-Au magnetic nanoparticles coated horseradish peroxidase (HRP) and graphene sheets (GS)-Nafion film modified screen-printed carbon electrode (SPCE) was fabricated. To construct the H2O2 biosensor, GS-Nafion solution was first dropped onto the surface of SPCE. Subsequently, the biocomposites of Fe3O4-Au magnetic nanoparticles coated HRP were adsorbed on the surface with the aid of an external magnetic field to fabricate the SPCE vertical bar GS-Nafion/Fe3O4-Au-HRP electrode. X-ray powder diffractometer (XRD), scanning electron microscopy (SEM), electrochemical impedance spectroscopy (EIS), cyclic voltammetry (CV) and chronoamperometry(i-t curve) were employed to study the synthesis of GS, the construction processes and electrochemical properties of the biosensor. Under optimized experimental conditions, CV demonstrated that the direct electron transfer (DET) of HRP was realized. The biosensor had an excellent performance in terms of electrocatalytic reduction toward H2O2. The linear response of the biosensor to H2O2 was in the concentration range of 2.0 x 10(-5) mol/L to 2.5 x 10(-3) mol/L (R = 0.9994) with a detection limit of 1.2 x 10(-5) mol/L (S/N = 3). The proposed electrochemical biosensor was sensitive, rapid, disposable with low cost, fewer sample volume, easy preparation and strong anti-interference, which showed great promise for screen-determination of trace H2O2 in real samples. (C) 2013 Elsevier Ltd. All rights reserved.