An electrochemical sensor for hydrogen peroxide (H₂O₂) detection was established using 1-aminopyrene (AP) as a linker between horseradish peroxidase (HRP) and reduced graphene oxides (rGO) modified glassy carbon electrode. The AP can combine with HRP via a covalent bond and combine withrGO via π-π conjugation. The covalent bond linkages can prevent the enzymes leaching of the enzymes and the π-π conjugate combination can facilitate electrons transfer, which plays synergistic effects to improve the performance of this sensor. The electrochemical activity of the HRP-AP/rGO was described by cyclic voltammetry, alternating current impedance and amperometric techniques. Under the optimal conditions, the biosensor showed a wide linear range from 1.5 μM to 28.5 μM and a low detection limit of 0.5 μM with good stability and high selectivity, confirming that the sensor is well-suited for the detection of H₂O₂ during biological processes.