The construction and performance of a novel colloidal gold-carbon nanotubes (CNT) composite electrode using Teflon as the non-conducting binding material, is reported. The resulting Au-coll-CNT-Teflon electrode shows significantly improved responses to hydrogen peroxide when compared with other carbon composite electrodes, including those based on CNTs. The incorporation of glucose oxidase (GOx) into the new composite matrix allowed the preparation of a mediatorless glucose biosensor. Once enzyme, CNT and colloidal gold loadings into the composite matrix were optimized, the analytical characteristics of the calibration graph for glucose, obtained by amperometry at +0.5 V, showed a remarkably higher sensitivity than that achieved with other GOx-CNT bioelectrodes. The calculated value of the apparent Michaelis-Menten constant, 14.9 mM, suggests a high affinity of the enzyme-substrate under the microenvironment provided by gold nanoparticles. Both repeatability of the amperometric measurements, reproducibility with different biosensors, lifetime and storage ability show a good analytical performance, which compares advantageously with previous GOx-CNT biosensor designs. The biosensor was applied for the rapid determination of glucose in beverages recommended for sport practice. (c) 2007 Elsevier B.V. All rights reserved.