Organic semiconductors and conducting polymers are the most promising next-generation conducting materials for electrochemical biosensors as the greener and cheaper alternative for electrodes based on transition metals or their oxides. Therefore, polycarbazole as the organic semiconducting polymer was electrochemically synthesized and deposited on working electrode. Structure and semiconducting properties of polycarbazole have theoretically and experimentally been analyzed and proved. For these electrochemical systems, a maximal sensitivity of 14 μA·cm−2·mM−1, a wide linear range of detection up to 5 mM, and a minimal limit of detection of around 0.2 mM were achieved. Moreover, Michaelis’s constant of these sensors depends not only on the enzyme but on the material of electrode and applied potential. The electrocatalytic mechanism and performance of the non- and enzymatic sensors based on this material as a conducting layer have been discussed by estimating pseudocapacitive and faradaic currents and by adding glucose as an analyte at the different applied potentials. In this work, the attention was focused on the electrochemical origin and mechanism involved in the non- and enzymatic oxidation and reduction of glucose.