This work describes the construction of a biosensor for glutathione disulfide (GSSG) based on a nanostructured platform composed by MWCNTs, chitosan (CHIT) and the redox mediator 3,5-dinitrobenzoic acid. The dispersion of MWCNTs and CHIT showed a good stability and was used to modify the glassy carbon electrode (GCE). The nanostructured platform was characterized by scanning electron microscopy (SEM) and electrochemical techniques. The R-NO/R-NHOH redox couple was electrogenerated in situ by cycling the potential between 0.2 and -0.4V vs. SCE. After activating the nanostructured platform, glutathione reductase was easily immobilized on the electrode surface by using glutaraldehyde as functional linker. The biosensor performance was optimized in terms of amount of enzyme, effect of CHIT concentration and NADH amount. Under optimized conditions, the biosensor response for GSSG sensing was linear from 2.0 up to 35 mu mol L-1 with detection and quantification limits achieving values of 0.6 and 2.0 mu mol L-1, respectively and sensitivity of 6.24 mA L mol(-1). The apparent Michaelis-Menten constant (K-M(app)) obtained by amperometry for the immobilized glutathione reductase on the nanostructured platform was 60 mu mol L-1. (C) 2012 Elsevier Ltd. All rights reserved.