In this work, firstly methylene blue (MB) was electropolymerized onto pencil graphite electrode (PGE) surface for the electrocatalytic oxidation of NADH. Cyclic voltammograms show that oxidation potential of NADH at Poly-MB/PGE shifted to negative direction about 300 mV compared with bare PGE. These results indicate that Poly-MB/PGE exhibits a good electrocatalytic activity toward NADH oxidation. Then, a glucose biosensor study was performed based on the determination of enzymatically generated NADH by glucose dehydrogenase (GDH) which immobilized onto Poly-MB/PGE using glutaraldehyde cross-linking procedure. The biosensing of glucose in flow injection analysis (FIA) system was performed at GDH/Poly-MB/PGE for the first time. The electrocatalytic oxidation currents of enzymatically produced NADH obtained from FI amperometric current-time curves recorded at + 200 mV and in phosphate buffer solution at pH 7.0 containing 1.0 M KCl were linearly related to the concentration of glucose. Linear calibration plots are obtained in the concentration range from 0.01 to 1.0 mM. The limit of detection (LOD) was found to be 4.0 mu M. A fast, sensitive, low-cost and disposable glucose biosensor was constructed in FIA system using GDH/Poly-MB/PGE; therefore, it might provide a new perspective for the fabrication of biosensor of other compounds such as glutamate, lactate and alcohol.