Electrochemical behavior of electro-generated poly-pyronin B (PyB) film was reported at a screen-printed carbon electrode (SPCE). The poly(PyB) modified SPCE showed excellent redox activity in neutral and alkaline media. Surface topography of poly(PyB) film modified electrode was analyzed by using atomic force microscopy (AFM) and scanning electron microscopy (SEM). Poly(PyB) film coated electrode was characterized by electrochemical impedance spectroscopy (EIS), UV–visible absorption spectroscopy (UV–vis) and cyclic voltammetry (CV). The poly(PyB) modified electrode showed electrocatalytic response to the reduced form of nicotinamide adenine dinucleotide (NADH) in physiological condition, and was used for the detection of NADH with high selectivity. The anodic peak current was linearly related to concentrations of NADH over the range from 110-5 M to 5.210-4 M, and the detection limit was 510-7 M (S/N = 3).