In-vitro on-line glucose monitoring is described, based on microdialysis sampling and amperometric detection operated in a flow-injection system. Samples were injected into a two-electrode microcell containing an Ag/AgCl quasi-reference electrode and a glucose enzyme electrode as the working electrode, operated at + 0.15 Vvs. Ag/AgCl. The enzyme electrode is constructed by mixing the ‘wired’ glucose oxidase into carbon paste. {Poly[1-vinylimidazole osmium(4,4′-dimethylbipyridine)₂Cl)]}^+/2+ was used to ‘wire’ the enzyme. The non-coated electrodes, cross-linked with poly(ethylene glycol) diglycidyl ether, responded linearly to glucose concentrations up to 60 mM, and were characterized by a sensitivity of 0.23 μA mM⁻¹ cm⁻², when operated in flow injection mode and of 5.4 μAmM ^–1 cm^–2 in steady-state conditions. This sensitivity of the resulting enzyme electrode was 50% lower than that of similarly prepared but non-cross-linked electrodes. However, the cross-linked electrodes showed superior operational and storage stabilities, which were further improved by coating the electrodes with a negatively charged Eastman AQ film. An in-house designed microdialysis probe, equipped with a polysulphone cylindrical dialysis membrane, yielded a relative recovery of 50–60% at a perfusion rate of 2.5 μl/min^–1 in a well stirred glucose solution. The on-line set up effectively rejected common interferences such as ascorbic acid and 4-acetaminophen when present at their physiological concentrations.