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 −0.2V to 0.6V vs. Ag|AgCl electrode. The glucose biosensor showed a high sensitivity of 14.24nAμM −1 cm −2 at a low applied potential (−0.1V vs. Ag|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 (0–18mM) and the biosensor current response. The results suggest that the biosensor was suitable for subcutaneous glucose monitoring by reverse iontophoresis.