Glucose oxidase (GOD) was immobilized in Bombyx mori silk fibroin membrane by only physical treatment, i.e., stretching without any chemical reagents. This is due to the structural transition of the silk fibroin membrane from random coil to antiparallel β‐sheet (Silk II) induced by the stretching treatment. Permeability coefficients of glucose and oxygen through the fibroin membrane were determined; the permeability of glucose decreased with increasing degree of stretching. The immobilized enzyme activity was characterized with apparent Michaelis constant K and maximal activity V_m. Optimum pH of the activity of the immobilized enzyme was shifted to the value around neutrality, and the activity was maintained to the higher values on both sides of the optimum pH compared with the case of free enzymes. Thermal stability was scarcely lost even at 50°C, although the free enzyme lost about 70% of the original activity. Thus, the stabilities of the enzyme vs. pH and heat were much improved by the immobilization with silk. Glucose sensor prepared with this GOD‐immobilized fibroin membrane was developed; the capabilities such as the response time, calibration curve, and repeating usage were determined.