An electrochemical biosensor for determination of hydrogen peroxide (H2O2) has been developed by the hybrid film of poly(methylene blue) and FAD (PMB/FAD). The PMB/FAD hybrid film was performed in PBS (pH 7) containing methylene blue and FAD by cyclic voltammetry. Repeatedly scanning potential range of −0.6–1.1 V, FAD was immobilized on the electrode surface by electrostatic interaction while methylene blue was electropolymerized on electrode surface. This modified electrode was found surface confined and pH dependence. It showed good electrocatalytic reduction for H2O2, KBrO3, KIO3, and NaClO as well as electrocatalytic oxidation for NADH. At an applied potential of −0.45 V vs. Ag/AgCl, the sensor showed a rapid and linear response to H2O2 over the range from 0.1 μM to 960 μM, with a detection limit of 0.1 μM and a significant sensitivity of 1109 μA mM−1 cm−2 (S/N = 3). It presented excellent stability at room temperature, with a variation of response current less than 5% over 30 days.