Electrochemical sensing of glucose on a gold electrode modified by the sequential incorporation of the mediator, 1,4-diaminoanthraquinone (DAAQ) and the enzyme, glucose oxidase (GOD), through covalent linkage onto a self-assembled monolayer configuration is described in this paper. It has been shown that two alternate layers of enzyme and mediator could be formed on the surface. The modified electrode is catalytic towards glucose oxidation. The catalytic response becomes more well behaved with the two-layer configuration and it is demonstrated by the amperometric experiments. The results also indicated that the mediator catalyzes the oxidation of H2O2, and also it establishes a weak electrical communication between the enzyme and the electrode surface. The results present the scope for the fabrication of membrane-free biosensors of ultra-thin dimensions. (C) 2002 Elsevier Science B.V. All rights reserved.