An electrochemical sensing platform was developed based on the integration of room-temperature ionic liquids (1-butyl-3-methylimidazolium tetrafluoroborate, BMIM.BF4) and multi-walled carbon nanotubes (MWNTs) with polymeric matrix (chitosan, CHIT). The resulting composite were investigated and characterized by FTIR, TEM, SEM, EDS and electrochemical methods. The BMIM.BF4/MWNTs/CHIT have good dispersibility in aqueous solution and can form a relative uniform film with unique structure. Electrochemical studies suggested that the BMIM.BF4/MWNTs/CHIT composite system provided a synergistic augmentation on the voltammetric and amperometric behaviors of electrochemical oxidation of NADH, which indicated by the decrease of the peak potential of NADH oxidation and the improvement of amperometric response. Additionally, the BMIM.BF4/MWNTs/CHIT/GC electrode shows good analytical performance such as low detection limit (0.06 mu M), good regeneration and antifouling properties for determination of NADH. This nanomaterials-based composite may be used as electrochemical transducers and have potential application for designing a variety of NAD(+)-dependent electrochemical biosensors. (c) 2007 Published by Elsevier Ltd.