The glucose oxidase (GO) enzyme was immobilized on chitosan-based porous composite membranes using a covalent bond between GO and the chitosan membrane. The chitosan-based porous membranes were prepared by the combination of the evaporation- and non-solvent-induced phase separation methods. To increase the membrane conductivity, carbon nanotubes (CNTs) were added to the chitosan solution. The resulting membranes were characterized in terms of water permeability, surface morphology and surface chemistry. Enzyme immobilization was performed on the chitosan membranes with and without activation using glutaraldehyde (GA). Three different configurations of working electrodes were evaluated to investigate the potential use of the modified membranes in biosensors. The results show that enzyme immobilization capacity was greater for membranes that had been activated than for membranes that had not been activated. In addition, activation increased the stability of the enzyme immobilization. The immobilization of GO x on chitosan-based membranes was influenced by both pH and the concentration of the enzyme solution. The presence of CNTs significantly increased the electrical conductivity of the chitosan membranes. The evaluation of three different configurations of working electrodes suggested that the third configuration, which was composed of an electrode-mediator-(chitosan and carbon nanotube) structure and enzyme, is the best candidate for biosensor applications. (C) 2013 Elsevier Inc. All rights reserved.