Enzyme electrodes were prepared by covalent immobilization of glucose oxidase (GOx) on the films of conducting copolymer obtained by electrochemical copolymerization of 3-methylthiophene and thiophene-3-acetic acid. Onto the enzyme electrodes, 2,5-dihydroxyphenyl (DHP) groups were introduced as redox mediators by the reaction of 2,5-dihydroxybenzaldehyde using alkylenediamines as linker molecules. The mediator-carrying enzyme electrodes were applied to reagentless glucose sensing system. It was found that response current to glucose was increased by introducing DHP groups, and the longer the alkylene chain of used alkylenediamine was, the higher the response current became. For a comparison, poly-L-lysine was linked onto the films of the conducting copolymer in advance, and immobilization of GOx and then introduction of DHP groups were carried out. The mediator-carrying electrode prepared thus gave higher amperometric response than those prepared with alkylenediamines, suggesting that amino groups Of poly-L-lysine functioned effectively as the sites for binding GOx and DHP groups. (c) 2005 Elsevier Ltd. All rights reserved.