The characteristic of Myoglobin as mimetic enzyme was studied and applied in the determination of H2O2 with o-phenylenediamine as substrate. The absorbance obtained at 40°C was found to be proportional to the concentration of H2O2 in the range of 2.0 × 10-5-2.0 × 10-7 mol/L, with a correlation coefficient of 0.9991. The proposed method is simple and highly sensitive to a detection limit of 2.7 × 108 mol/L. The relative standard deviations were within 5% for the determination of different concentrations of H2O2. Satisfied results were obtained in the determination of H2O2 in rainwater by this method, and the recoveries were at the range of 97.2%-105.4%.