Here, we report on a novel, versatile approach for the preparation of mediated enzyme electrodes, demonstrated using cross-linked films of glucose oxidase and a range of functionalised osmium complexes on graphite electrodes. Response of enzyme electrodes are optimised by evaluation of glucose response as a function of variation in ratios of [Os(2,2'-bipyridine)2(4-aminomethyl pyridine)Cl](+) redox mediator, polyallylamine support and glucose oxidase enzyme cross-linked using a di-epoxide reagent in films on graphite. Lowering of the redox potential required to mediate glucose oxidation is achieved by synthesis of complexes using (4,4'-dimethyl-2,2'-bipyridine) or (4,4'-dimethoxy-2,2'-bipyridine) as a ligand instead of (2,2'-bipyridine). Enzyme electrodes prepared using the complexes based on dimethoxy- or dimethyl-substituted bipyridines provide glucose oxidation current densities of 30 and 70 μA cm(-2) at 0.2 and 0.35 V applied potential compared to 120 μA cm(-2) at 0.45 V for the initial enzyme electrode, under pseudo-physiological conditions in 5 mM glucose, with stability of signals proving inadequate for long-term operation. Current output and stability may be improved by selection of alternate anchoring and cross-linking methodology, to provide enzyme electrodes capable for application to long-term glucose biosensors and anodes in enzymatic fuel cells.