This study investigated the development and optimization of a flexible printed circuit board-based glucose biosensor with an emphasis on high sensitivity, selectivity, and overall performance. Advances in glucose biosensing have highlighted its importance in medical diagnostics, especially diabetes management. The fabrication process involves depositing a RuO2 sensing film on a flexible printed circuit board (FPCB) by radio frequency sputtering. Enzyme-based modification using glucose oxidase (GOx), (3-aminopropyl) triethoxysilane (APTES), and glutaraldehyde (GA) to enhance selectivity and catalytic reactions. And through Scanning Electron Microscopy and electrochemical impedance spectroscopy, the sensing film, and the effect of modification on the charge transfer rate and performance improvement were analyzed. This glucose biosensor has excellent linearity, sensitivity, and reproducibility. The study also assessed response time and selectivity. The response time efficiency of the biosensor solidified its utility in point-of-care monitoring, while selectivity experiments validated its ability to distinguish glucose from interfering substances, ensuring accuracy in practical applications. According to the experimental results, the enzymatic glucose biosensor has the best average sensitivity and linearity of 44.42 mV/mM and 0.999 with a response time of 6 seconds.