Semiconductor‐based biosensors are suitable for the detection of small molecules such as glucose as long as they have molecular charges. However, biological samples include various ionic molecules such as proteins; thus, their nonspecific adsorption should be prevented to reduce background noise. In this study, we optimized a field‐effect transistor (FET)‐based glucose sensor, that is, a glucose transistor, for more precise and highly sensitive glucose sensing. Using the glucose transistor modified with 4‐mercaptophenylboronic acid (PBA) on a gold gate electrode, we found that the detection sensitivity to glucose was increased to approximately 60 mV/decade from about 40 μM. This means that the PBA on the gate induced a diol‐binding site with glucose through an equilibrium reaction, resulting in an increase in the density of negative charges in the PBA‐glucose conjugate. Moreover, an oligoethyleneglycol (OEG) monolayer was coated on the gate as a molecular sieve interface so that the PBA molecules were embowered in the OEG membrane, but low‐molecular‐weight glucose could pass between the OEG molecules and reach the PBA molecules at the gate. The detection sensitivity of the biosensor with the optimized glucose transistor to glucose remained, to some extent, even in a solution including albumin. Thus, a platform based on the optimized glucose transistor is available for the practical use of a novel sensor that can detect glucose even at the low concentrations included in biological fluids.