In this work, two methods of glucose oxidase (GOD) immobilization for peroxide based glucose biosensor had been investigated. For the first immobilization method, GOD was immobilized in freeze–thawed poly (vinyl alcohol) (PVA) at three different PVA concentrations (5%, 10% and 15% PVA). The higher the PVA concentration used for immobilization, the better the retention of the enzyme in the membrane. Nevertheless, higher PVA concentration did not necessarily correlate well with enzyme activity. Freeze–thawed PVA–GOD membranes with 10% PVA showed the highest sensitivity to glucose. The performances of freeze–thawed PVA–GOD membranes with 15% and 5% PVA was comparable. For the second immobilization method, GOD was immobilized within PVA/tetramethoxysilane (TMOS) matrix with different (3–glycidoxypropyldimenthylethoxy) silane (3GPDES) concentrations. For the membranes prepared with 1:1 (TMOS: 3GPDES) volumetric ratio, the percentage of enzyme activity remaining at day 40 was about 51%. Meanwhile, membrane prepared with 1:2 (TMOS: 3GPDES) and 1:3 (TMOS: 3GPDES) were quite stable with 69% and 58% of enzyme activity remaining at day 40, respectively. Overall, the glucose biosensor showed very promising performances.