The amperometric bienzyme glucose biosensor utilizing horseradish peroxidase (HRP) and glucose oxidase (GOx) immobilized in poly(toluidine blue O) (PTBO) film was constructed on multi-walled carbon nanotube (MWNT) modified glassy carbon electrode. The H RP layer could be used to analyze hydrogen peroxide with toluidine blue O(TBO) mediators, while the bienzyme system (HRP + GOx) could be utilized for glucose determination. Glucose underwent biocatalytic oxidation by GOx in the presence of oxygen to yield H2O2 which was further reduced by HRP at the MWNT-modified electrode with TBO mediators. In the absence of oxygen, glucose oxidation proceeded with electron transfer between GOx and the electrode mediated by TBO moieties without H2O2 production. The bienzyme electrode offered high sensitivity for amperometric determination of glucose at low potential, displaying Michaelis-Menten kinetics. The bienzyme glucose biosensor displayed linear response from 0.1 to 1.2 mM with a sensitivity of 113 mA M-1 cm(-2) Stan applied potential of -0.10 V in air-saturated electrolytes. (C) 2010 Elsevier Ltd. All rights reserved.