A glucose microsensor with a nanoscale conducting matrix and redox mediator has been developed and results of a detailed in vitro and ex vivo study of glucose consumption are presented. This microsensor is a conducting polymeric glucose monitoring device and is based on an enzymatic detection technique. A multilayer multipolymeric fabrication approach using biocompatible materials was investigated to construct such a microsensor device. A novel two-electrode electrochemical sensing cell consisting of a modified platinum working electrode (WE)with selective deposition of a conducting polymer polypyrrole (PPy) and a plain platinum (pseudo-reference/counter) electrode has been used. As a redox mediator, the ferrocene molecule has been successfully immobilized in the nanoscale PPy matrix that is deposited onto the surface of the WE. The ferrocene redox mediator lowers the operational potential of the sensor and helps avoid the introduction of noise and oxidation of interference molecules such as ascorbic acid and uric acid. Good sensitivity (similar to 20 nA/mM of glucose) and selectivity for glucose were accomplished. Detailed structural, in vitro and ex vivo properties as well as calibration results of the microsensor device are presented. Published by Elsevier Ltd.