The modification of glassy carbon (GC) electrode with deoxyribonucleic acid (DNA) and graphene is utilized as a new efficient platform for entrapment of methylene blue (MB). Electrochemical and electroanalytical properties of the modified electrode (DNA/graphene/MB) were investigated by cyclic voltammetry (CV), electrochemical impedance spectroscopy (EIS) and amperometry techniques. Cyclic voltammetric results indicated the excellent electrocatalytic activity of the resulting electrode toward oxidation of beta-nicotinamide adenine dinucleotide (NADH) at reduced overpotential (0.1 V vs. Ag/AgCl). It has been found that the DNA/graphene/MB modification has significantly enhanced the effective electrode response toward NADH oxidation. Cyclic voltammety and rotating disk electrode (RDE) experiments indicated that the NADH oxidation reaction involves two electrons and an electrocatalytic rate constant (k(obs)) of 1.75 x 10(6) mol(-1) L s(-1). The electrochemical sensor presented better performance in 0.1 mol L-1 phosphate buffer at pH 7.0. Other experimental parameters, such as the DNA, graphene, MB concentrations and the applied potential were optimized. Under optimized conditions, a linear response range from 10 mu mol L-1 to 1.50 mmol L-1 was obtained with a sensitivity of 12.75 mu A L mu mol(-1). The detection and quantification limits for NADH determination were 1.0 mu mol L-1 and 3.3 mu mol L-1, respectively. (C) 2013 Elsevier Ltd. All rights reserved.