The enzyme catalytic reaction properties of H2O2-4-aminoantipyrine (4-AAP)-the different chlorophenolic derivatives color systems using hemoglobin as enzyme mimetic of peroxidase was studied. Meanwhile, the relationship of structure-effect for the different chlorophenolic derivatives as substrate has been discussed, and the mechanism of catalytic reaction has also been investigated. The method for the determination of hydrogen peroxide was proposed using 4-AAP-2,3,4-trichlorophenol system with hemoglobin as catalyst. The sensitivity of method proposed for the determination of H2O2 was better than other similar systems. The apparent molar absorptivity of the chromogenic reaction for H2O2 was 2.21×104 L*mol-1*cm-1. Satisfied results were obtained in the determination of H2O2 in synthetic samples by this method. Also, the method was coupled with glucose oxidation reaction for the determination of glucose in human serum.