A poly-o-phenylenediamine film modified glassy carbon electrode (PoPD/GC) was successfully prepared by cyclic voltammetry in acetate buffer solution. The polymerization mechanism of oPD is discussed. The impedance behavior and morphology of the PoPD membrane were characterized using cyclic voltammetry, electrochemical impedance spectroscopy, and scanning electron microscopy, respectively. It is discovered that the PoPD/GC prepared only in acetate buffer solution had dual electrocatalytic activity toward the oxidation and reduction of H2O2. The optimal buffer solution pH, scanning rate, monomer concentration, and number of scannings for film forming were 4.2, 0.05 V s−1, 6.0 mmol L−1, and 30, respectively. The linear ranges between the anodic (Δia) or cathodic (Δic) current and H2O2 concentration were 0.07−1.0 × 104 and 0.04−4.5 × 104 μmol L−1, respectively. The corresponding calibration curves were Δia (μA) = 8.03c (mmol L−1) + 6.36 (n = 18, R2 = 0.9989) and Δic (μA) = −5.52c (mmol L−1) − 0.77 (n = 18, R2 = 0.9990) with a detection limit of 0.03 and 0.02 μmol L−1 (S/N = 3), respectively. The PoPD/GC prepared in the optimal conditions showed good stability and quick response (<0.2 s) to H2O2, which was successfully applied to the determination of H2O2 in real water samples with satisfactory results.