A biosensor based on hemoglobin-Fe3O4@SiO2 nanoparticle bioconjunctions modified indium-tin-oxide (Hb/Fe3O4@SiO2/ITO) electrode was fabricated to determine the concentration of H2O2. UV-vis absorption spectra, fourier transform infrared (FT-IR) spectroscopy, cyclic voltammetry (CV) and high-resolution transmission electron microscopy (HRTEM) were used to characterize the bioconjunction of Fe3O4@SiO2 with Hb. Experimental results demonstrate that the immobilized Hb on the Fe3O4@SiO2 matrix retained its native structure well. In addition, Fe3O4@SiO2 nanoparticles (NPs) are very effective in facilitating electron transfer of the immobilized enzyme, which can be attributed to the unique nanostructure and larger surface area of the Fe3O4@SiO2 NPs. The biosensor displayed good performance for the detection of H2O2 with a wide linear range from 2.03×10−6 to 4.05×10−3 mol/L and a detection limit of 0.32 µmol/L. The resulting biosensor exhibited fast amperometric response, good stability, reproducibility, and selectivity to H2O2.