The IR spectra, the electronic absorption bands, and scanning electron microscopy (SEM) of the blend membranes of regenerated silk fibroin and glucose oxidase were reported for the first time and the second generation of glucose sensor based on glucose oxidase immobilized in the regenerated silk fibroin membrane was first constructed. The IR absorption spectra of the pure regenerated silk fibroin membrane were ascribed to its structural characteristics by which the part transition from silk I to silk II was recognized in ethanol immersion. Those spectra of the blend membrane of regenerated silk fibroin and glucose oxidase were identified as a composite of the absorption bands characteristic of both macromolecules. The electronic absorption bands showed that the glucose oxidase in the membrane exists in aggregates. A sea islands’ structure was observed by SEM. These findings suggest that the regenerated silk fibroin and glucose oxidase are incompatible and their molecular interactions are very weak. A tetrathiafulvalene-mediating glucose sensor, employing immobilization of glucose oxidase by regenerated silk fibroin, was fabricated. The influences of temperature, applied potential, and pH on steady-state electrocatalytic oxidation of glucose at the sensor were evaluated. The response of the sensor to glucose under N-2 saturation reached 95% steady-state current within 40 s. The sensor could be used repeatedly for 1.5 months without deterioration of the response.