A simple and practical sensor of hydrogen peroxide (H2O2) was designed successfully. The mixture of horseradish peroxidase (HRP) and chitosan (Chit) are effectively immobilized on the surface of poly-L-leucine/polydopamine modified glassy carbon electrode (PL-LEU/PDA/GCE). Under the optimum conditions, the biosensor based on HRP exhibits a fast amperometric response (within 3 s) to H2O2. The linear response range of the sensor is 0.5–952.0 μmol L–1, with the detection limit of 0.1 μmol L–1 (S/N = 3) and the sensitivity of 0.23 A L moL–1 cm–2. The apparent Michaelis–Menten constant (k M app) of the biosensor is evaluated to be 0.12 mmol L–1, which suggests that the HRP-Chit/PL-LEU/PDA/GCE shows a higher affinity for H2O2. The sensor exhibits good sensitivity, selectivity, stability and reproducibility. The proposed method has been successfully applied to the determination of H2O2 in practical samples.