A sol-gel material of (3-mercaptopropyl) trimethoxysilane (MPTMS) is proposed to function as permselective membranes of biosensors. Permselectivity of MPTMS and Nafion was compared by studying their anti-interferent ability. Membrane porosity of MPTMS and Nafion was first confirmed via voltammetric responses in ferrocynite/ferricynite solution. In the comparison studies, membranes prepared with 20% MPTMS in phosphate buffer solution (PBS) and 1% Nafion in 2-propanol (IPA) were used as coating materials on the surface of two platinum (Pt) electrodes. These electrodes were used to electrochemically measure the response currents of ascorbic acid, uric acid, and acetaminophen. The results indicate that the MPTMS-based electrode produced much less response currents from the interference species compared to that of the Nafion-based electrode. This denotes that the anti-interferent ability ofMPTMS is superior to that of Nafion. A platinum working electrode containing glucose oxidase (GOx) immobilized by poly-aniline (PA) and then modified by MPTMS was developed and evaluated. The results show that the optimum applied potential for the glucose biosensor is 0.4 V. This operational potential not only inhibits the response currents from ascorbic acid, uric acid, and acetaminophen but also produces rather high signals for glucose.