The biosensor was constructed for determination of glucose by using glucose oxidase enzyme immobilized on poly(thiophene-3-boronic acid) (PTBA). Boronic acid functionalized polythiophene layer was obtained by electrochemical polymerization of Thiophene (Th) and thiophene-3-boronic acid (TBA) with different monomer rations. The reconstitution of the apo-glucose oxidase (apo-GOx) on a complexed flavin adenine dinucleotide (FAD) linked to polythiophene boronic acid (PTBA) monolayer yields an electrically contacted enzyme monolayer. The GOx-reconstituted enzyme electrode exhibited excellent electrocatalytic activities toward the reduction and oxidation of hydrogen peroxide as well. The PTBA/FAD/GOx biosensor shows an excellent performance for glucose at +0.4 V with a high sensitivity (2.14 mu A/mM) and lower response time (similar to 5 s) in a wide concentration range of 0.5-18 mM (correlation coefficient of 0.9952). Furthermore, the effects of applied potential, pH, temperature, electroactive interference, stability and reusability of the biosensors were discussed. (c) 2013 Elsevier B.V. All rights reserved.