A continuous, noninvasive glucose monitoring method is necessary for diabetic patients to reduce the risk of hypoglycemia. This paper was to develop a bifunctional biosensor for the simultaneous extracting and detecting the subcutaneous glucose by reverse iontophoresis. The biosensor was constructed by a tri-electrode planar configuration and multi-layer sensitive membrane composed of mediator, enzyme and cross linking agents. A hydrogel membrane was deposited on the top of the device to act as a collected reservoir of the extracted glucose. The electrochemical characteristic of the amperometric biosensor was evaluated using cyclic voltammetry at applied potentials from -0.2 V to 0.6 V vs. Ag vertical bar AgCl electrode. The glucose biosensor showed a high sensitivity of 14.24 nA mu M(-1) cm(-2) at a low applied potential (-0.1 V vs. Ag vertical bar AgCl). Finally, the biosensor was used for noninvasive glucose monitoring by reverse iontophoresis using the naked mouse skin as the model. An excellent linear relationship (r(2) = 0.99) was found between the subcutaneous glucose concentration (0-18 mM) and the biosensor current response. The results suggest that the biosensor was suitable for subcutaneous glucose monitoring by reverse iontophoresis. (C) 2011 Elsevier B.V. All rights reserved.