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 HzO2 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.