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An electrochemical method for the preparation of poly(pyronin B) film was proposed in this paper. A poly(pyronin B) (poly(PyB)) film modified glassy carbon electrode (GCE) has been fabricated via an electrochemical oxidation procedure and applied to the electrocatalytic oxidation of reduced form of nicotinamide adenine dinucleotide (NADH). The poly(PyB) film modified electrode surface has been characterized by atomic force microscope (AFM), scanning electron microscope (SEM), electrochemical impedance spectroscopy (EIS), UV-visible absorption spectrophotometry (UV-vis) and cyclic voltammetry (CV). These studies have been used to investigate the poly(PyB) film, which demonstrates the formation of the polymer film and the excellent electroactivity of poly(PyB) in neutral and even in alkaline media. Due to its potent catalytic effects towards the electrooxidation of NADH at lower potential (0.0?V), poly(PyB) film modified electrode can be used for the selective determination of NADH in real samples because of dopamine, ascorbic acid and uric acid oxidation can be avoided at this potential. The catalytic peak currents are linearly dependent on the concentrations of NADH in the range of 1.0×10^?6 to 5.0×10^?4?mol/L with correlation coefficients of 0.999. The detection limits for NADH is 0.5×10^?6?mol/L. Poly(PyB) modified electrode also shows good stability and reproducibility due to the irreversible attachment of polymer film at GCE surface.