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A novel amperometric hydrogen peroxide sensor was proposed by co-immobilizing new methylene blue (NMB) and Horseradish peroxidase (HRP) on glassy carbon electrode through covalent binding. The electrochemical behavior of the sensor was studied extensively in 0.1 mol/L phosphate buffering solution (pH = 7.0). The experiments showed NMB could effectively transfer electrons between hydrogen peroxide and glassy carbon electrode. The electron transfer coefficient and apparent reaction rate constant were determined to be 0.861 and 1.27 s−1. The kinetic characteristics and responses of sensor on H2O2 were investigated. The Michaelis constant is 8.27 mol/L and the linear dependence of current on H2O2 is in the range of 2.5–100 μmol/L. At the same time, the effects of solution pH, buffer capacity, and temperature on the sensor were examined.