A facile and efficient synthesis of hemin containing metal-organic framework (MOF) nanocomposite was reported. Two dimensional (2D) Ni-MOF nanosheets have been used as a new type of matrix material to immobilize hemin. Scanning electron microscopy (SEM), energy dispersive X-ray spectroscopy (EDS)-mapping, thermal gravity analysis (TGA) suggest the hemin is uniformly distributed in the Ni-MOF nanosheets. Ultraviolet-visible spectroscopy (UV-vis) analysis shows that hemin molecules are combined with MOFs nanosheets through pi-pi interaction. Electrochemical study indicates the Ni-MOFs/Hemin nanocomposite exhibits high electro-catalytic activities for H2O2 reduction and the biosensor offers high performance with a low detection limit of 0.2 mu M and a wide linear range of 1 mu M-0.4 mM. Furthermore, the Ni-MOFs/Hemin nanocomposite based biosensor displays high selectivity and stability owing to the protective effect of the 2D MOF nanosheets. More importantly, the biosensor was used to determine the H2O2 levels in human serum and disinfectant samples with satisfactory results.