Regioregular poly-3-hexylthiophene (regP3HT) and dithiobissuccinimidyl propionate (DTSP) have been used to prepare bifunctionalized gold nanoparticles (Bf AuNPs). Processable properties of regP3HT have been used to obtain regP3HT-AuNPs-DTSP film on a gold-coated glass surface ansd the succinimidyl group of DTSP in this regP3HT-AuNPs-DTSP/Au electrode has been utilized for covalent immobilization of glucose oxidase (GOx). The UV-visible (UV-vis), scanning electron microscopy (SEM), and Fourier transform infrared (FTIR) spectroscopic studies have been used to characterize regP3HT-AuNPs-DTSP/Au and Gox-regP3HT-AuNPs-DTSP/Au electrode, respectively. This GOx-regP3HT-AuNPs-DTSP/Au bioelectrode shows response time of 10 s, linearity from 25 to 300 mg/dL of glucose and the value of Michaelis-Menten constant as 5.85 mM (105.3 mg/dL). (c) Wiley Periodicals, Inc.