The activation of 3,5-dinitrobenzoic acid (3,5-DNBA) in situ is proposed for electrocatalytic detection of NADH at low overpotentials. Thus, 3,5-DNBA was strategically connected to a nanostructured surface based on MWCNTs and PEI (an amine rich polymer). Then, R-NO/R-NHOH was electrogenerated in situ by cycling the potential between 0.15 and -0.55 V versus SCE. The formation of an intermediate charge transfer complex is proposed for the reaction between NADH and redox mediator. The apparent Michaelis-Menten constant (K-m) obtained by RDE voltammetry and amperometry was in good agreement, around 10(-5) mol L-1. The k(obs) for the catalytic reaction evaluated by chronoamperometry and RDE voltammetry, was in good agreement and was found to be around 10(4) L mol(-1) s(-1). The sensor provides a linear response range for NADH from 4.0 up to 42.0 mu mol L-1 with a sensitivity of 71 nA L mu mol(-1), detection and quantification limits of 1.2 and 4.0 mu mol L-1 respectively, with a response time of 0.16s. (C) 2009 Elsevier Ltd. All rights reserved.