Glucose sensors are essential for managing diabetes, a metabolic disease affecting 1 in 10 adults globally. Enzyme-based biosensors, particularly those utilizing oxidoreductases, offer high specificity for glucose detection. This study explores the use of flavin-dependent glucose dehydrogenase from Aspergillus oryzae (AoGDH) in developing glucose sensors integrated into organic electrochemical transistors (OECTs) without mediators. We employed tri-thiophene monomer units to form conductive polymers interfacing with AoGDH, allowing sensing due to the proximity of the FAD cofactor. Despite AoGDH’s lower stability compared to glucose oxidase (GOx), its ability to function without oxygen sensitivity makes it advantageous. Using electropolymerization, we successfully incorporated AoGDH into the OECT gate electrode, demonstrating glucose detection in physiological ranges, albeit in buffer solutions. Furthermore, integrating this system into a 3D-printed capillary-driven microfluidic device facilitated on-demand sensor fabrication, enhancing portability and point-of-care application potential. This study underscores the viability of AoGDH-based, and ad hoc fabricated, OECT sensors for accurate and responsive glucose monitoring in biomedical applications.