A novel biosensor based on a myoglobin/gold nanoparticles/carbon spheres (Mb-AuNPs-CNs) 3-D architecture bioconjunction has been fabricated for the determination of hydrogen peroxide (H2O2). Cyclic voltammetry (CV), Fourier transform infrared (FT-IR) spectroscopy and scanning electron microscopy (SEM) were used to characterize the bioconjunction of the AuNPs-CNs with Mb. Experimental results demonstrate that the AuNPs-CNs hybrid material is more effective in facilitating electron transfer of the immobilized enzyme than CNs alone, which can be attributed to the unique nanostructure and larger surface area of the bioconjunction. The biosensor displayed good performance for the detection of H2O2 with a wide linear range from 0.28 μmol/L to 116.5 μmol/L and a detection limit of 0.12 μmol/L. The Michaelis-Menten constant K M app value was estimated to be 0.3 mmol/L. The resulting biosensor exhibited fast amperometric response, and good stability, reproducibility, and selectivity to H2O2.