The structural changes of the regenerated silk fibroin membranes for immobilizing glucose oxidase were characterized with FT-IR spectra. The results of electronic absorption spectra and scanning electron microscopy illustrated that the glucose oxidase in the membranes existed in aggretates. The blend membranes exhibited an island-like structure, attributable to the incompatible and weak intermolecular interactions between regenerated silk fibroin and glucose oxidase. The glucose sensor was constructed by combining glucose oxidase immobilized in regenerated silk fibroin membrane with an Eastman-AQ-ferrocene modified electrode. The advantages of the mediator include high stability and the ability to use a less positive potential for increased selectivity.