A highly active and stable composite of hemin (HN) supported by reduced graphene oxide/gold nanoparticles (HN-RGO/AuNPs) was prepared by one-pot hydrothermal method. The physicochemical properties of the as-prepared composites were characterized by field emission scanning electron microscopy (FESEM), transmission electron microscopy (TEM), UV-vis spectroscopy, Raman spectroscopy and X-ray diffraction technique (XRD). The HN-RGO/AuNP-modified electrode shows a stable and well-defined, surface-confined redox couple at an apparent formal potential of -0.317 V vs. Ag|AgCl with a surface coverage value of 2.239 × 10-10 mol cm-2. Compared with HN, HN-GO and HN-RGO, the HN-RGO/AuNP-modified electrode exhibits excellent electrocatalytic activity towards hydrogen peroxide (H2O2). Under optimum conditions, the HN-RGO/AuNP-modified electrode shows a wide linear response ranges from 0.05 μM to 518.15 μM towards H2O2 with a fast response time (3 s). The calculated sensitivity and limit of detection (LOD) of the biosensor were 3.99 μA μM-1 cm-2 and 16 nM, respectively. In addition, the Michaelis-Menten constant value of the biosensor is 0.13 mM, which indicates the high affinity of HN towards the reduction of H2O2. The proposed biosensor displays high sensitivity and selectivity towards H2O2 in the presence of common biologically co-existing species. The biosensor shows an acceptable practical ability in human serum, contact lens solution and milk samples with an appreciable recovery.