Ascorbate oxidase (AOx) was chemisorbed on glassy carbon modified with Au nanoparticles. Direct electron transfer (DET) between the copper sites of the immobilized enzyme and the electrode surface was examined with voltammetry. The dependence of the T1 site current maxima on the scan rate was found linear, suggesting a redox process controlled by surface chemistry with the anodic peak potential shifting negatively with the pH over the range from 5.6 to 7.0. The bioelectrocatalytic oxidation of the enzyme substrate L-ascorbate was observed under both aerobic and anaerobic conditions with a current response being more than 3.5 times higher in absence of oxygen than in its presence. Inhibition studies confirmed that the L-ascorbate oxidation results from the bioelectrocatalytic activity of the immobilized AOx. Amperometry carried out at pH = 7.0 and 298 K at a constant potential as low as 150 mV gave the following operational parameters: a detection limit of 1.5 mu M; linear dynamic range up to 4.8 mM and a sensitivity of 2.56 mu A mM(-1) mm(-2) (4.03 mu A mM(-1) mm(-2) at 200 mV). Under these conditions a real sample was assayed with a recovery between 95 and 102%. The enzyme electrode preserved up to 63% of its initial activity after 8 months storage. (C) 2013 The Electrochemical Society. All rights reserved.