A third-generation horseradish peroxidase (HRP) biosensor has been developed by adsorbing HRP on multi-wall carbon nanotube (MWNTs) monolayer modified gold electrode surface. The assembly process was investigated by electrochemical and spectroscopic techniques.Results showed that the immobilized HRP exhibited direct electrochemical behavior toward the reduction of H2O2. The resulting biosensor shows a fast amperometric response (＜2 s) to H2O2.The linear response range was from 5.0×10-7～1.0×10-5 mol/L with a detection limit of1.0×10-7mol/L. Moreover, the biosensor has a good reproducibility, and long-term stability.