A poly(N-acetylaniline)–Prussian blue (PNAANI–PB) hybrid composite film was prepared by co-electrodeposition. Using a spontaneous redox reaction, the PNAANI–PB hybrid film was prepared electrochemically by cyclic voltammetry in an electroplating bath containing a low concentration of N-acetylaniline. Its surface morphology showed that the PB nanoparticles in the hybrid film had a smaller size than those in the pure PB film. As a result of electrostatic interaction between the PNAANI and the PB particles, the PNAANI–PB hybrid film displays impressive synergistic effects that remarkably enhance its sensitivity, expand its linear range and broaden its acidic adaptability in the detection of H₂O₂. The hybrid film displays good stability in neutral solution, in contrast to the pure PB film, with a linear range from 10⁻⁶ to 10⁻³ M and a high sensitivity of 507.29 μA mM⁻¹ cm⁻² for H₂O₂ detection. An amperometric glucose biosensor was constructed by immobilizing glucose oxidase on the hybrid film. The biosensor showed an excellent response to glucose in the linear range 10⁻⁵–10⁻³ M and had a high sensitivity of 62.45 μA mM⁻¹ cm⁻². The glucose biosensor had a rapid response, good reproducibility and long-term stability and was free of interference from other coexisting electroactive species.