NAD(+)-dependent enzymes require the presence of catalysts for cofactor regeneration in order to be employed in enzymatic biofuel cells. Poly-(methylene green) catalysts have proven to help the oxidation reaction of NADH allowing for the use of such enzymes in electrocatalytic oxidation reactions. In this paper we present the development of 3D anode based on NAD(+)-dependent malate dehydrogenase. The 3D material chosen was reticulated vitreous carbon (RVC) which was modified with poly-(MG) for NADH oxidation and it also accommodated the porous immobilization matrix for MDH consisting of MWCNTs embedded in chitosan: allowing for mass transport of the substrate to the electrode. Scanning electron microscopy was used in order to characterize the poly-(MG)-modified RVC, and electrochemical evaluation of the anode was performed. (C) 2010 Elsevier Ltd. All rights reserved.