We report a glucose biosensor based on glucose oxidase (GOD) immobilized on TbY_xO_y sensing film deposited on the Si substrate through reactive cosputtering in this study. The structural properties of TbY_xO_y sensing films annealed at different temperatures (600, 700, 800, and 900 °C) were carried out using X-ray photoelectron spectroscopy and atomic force microscopy to obtain the optimal annealing condition. The TbY_xO_y electrolyte-insulator-semiconductor (EIS) sensor annealed at 900°C exhibited a higher sensitivity of 59.79 mV/pH, a lower drift rate of 0.26 mV/h, and a smaller hysteresis voltage of 1 mV in comparison with the other annealing temperatures. We attribute this behavior to the formation of a honeycomb-like structure and the reduction of lattice defects to improve the TbY_xO_y stoichiometry. Moreover, we compared two surface modification techniques with 3-aminopropyltriethoxysilane (APTES) and APTES+glutaraldehyde (GA) to immobilize the TbY_xO_y film surface and thus the enzymatic GOD was covalently bounded to the functionalized surface of a TbY_xO_y EIS sensor. The TbY_xO_y glucose biosensor prepared with APTES+GA has a high sensitivity (19.85 mV/mM) in solutions containing glucose at concentrations in the range 2–8 mM. This TbY_xO_y EIS biosensor is very promising sensing membrane materials for general clinical examination of blood glucose.