Abstract：Immobilizations of the graphene-based materials and enzyme are very important to electrochemical properties and use of the biosensor. In this work, a 0.005 mg/mL of graphite oxide and 0.005 mM of chlorauric acid were in sequence electrodeposited on the surface of gold electrode with potentiostatic elec-trolysis. After above procedure was repeated for 20 cycles, 2,5-di-(2-thienyl)-1-pyrrole-1-(p-benzoic acid) was electropolymerized on the modified electrode by cyclic voltammetry and finally formed functional conducting polymer film containing carbonyl groups on surface of the graphene/gold nanocomposite. To prepare hydrogen peroxide biosensor, the horseradish peroxidase was subsequently connected covalently to the film with DHC/NHS as activator. Research results indicated that the graphene/gold nanocomposite obtained using alternating electro-deposition has excellent dispersivity. The biosensor based on the material offers remarkable catalysis performance to the redox of hydrogen peroxide on the electrode surface. Current response of the sensor increases linearly with the increasing concentration of hydrogen peroxide over the range from 2 nM to 200 nM, with a correlation coefficient (R2) of 0.9996. The detection limit was found to be 0.67 nM (S/N=3). The sensitivity is more than other sensor reported in the literatures. In addition, covalent immobilization of the enzyme results in increasing the stability and reproducibility of the sensor. The relative standard deviation is 1.2 % for determination of 5 nM hydrogen peroxide for 20 times. After the sensor was stored at 4℃ for three months, its change value of the response is lower than 3%. The proposed method has been successfully applied to detect trace hydrogen peroxide in milk sample.