Abstract: In order to construct a novel hydrogen peroxide biosensor, hemoglobin was adsorbed on gold nanoparticles with polypyrrole membrane as the substrate. The modified electrodes were characterized by SEM, AFM, XPS, CV and EIS, and the sensor was used for the detection of hydrogen peroxide in milk.The results showed that materials were successfully modified to the electrode surface, and the size of the gold nanoparticles was about 15 nm. The best preparation conditions for Hb/AuNPs/Ppy/GCE were the pyrrole polymerization power 3.0×10^-3 C, the pH of PBS solution was 6.5, and the pH of supporting electrolyte solution was 7.0, the detection limit of hydrogen peroxide was 0.2 μmol/L (S/N=3), and the detection time was 12 s. In addition, the constructed sensor presented good stability and selectivity, the larger specific surface area of gold nanoparticles provided more sites to immobilize hemoglobin, and the hemoglobin and gold nanoparticles possessed a good catalytic effect on hydrogen peroxide.The standard recovery rate of hydrogen peroxide in milk was 92.7%~116.0%. Those results showed that Hb/AuNPs/Ppy/GCE is a promising electrochemical biosensor.