A conducting polymer of poly 3,4-ethylenedioxythiophene (PEDT) was used as a matrix for entrapment of enzymes onto a platinum electrode surface in order to construct amperometric biosensors. Glucose oxidase (GOD) was used as an example, and it was entrapped in the polymer during the electrochemical polymerization. Glucose in oxygenated solutions was tested by amperometric measurements at +650 mV (vs. SCE) in a batch system. The influence of several experimental parameters in the electropolymerization process was explored to optimize the analytical performance. The detection limit and sensitivity for this biosensor were 4×10−5 M and 15.2 mA M−1cm−2, respectively. A linear range of response was found from 0.2 to 8 mM of glucose. The response time was 2–5 s. The stability of the electropolymerized films was evaluated in operational conditions. The glucose probe, stored in buffer at 4 °C when not in use, showed a residual activity of 40% after about 1 month. Glucose in synthetic serum was determined under flow injection conditions using an amperometric flow cell.