Before or after the blend membrane of regenerated silk fibroin (RSF), poly(vinyl alcohol) (PVA), and peroxidase is treated with ethanol, RSF, PVA, and peroxidase maintain their own structures. The conformational transition of RSF in the blend membrane is accomplished from the silk I structure to the silk II structure by ethanol treatment, which is used to immobilize peroxidase. A ferrocene-mediating sensor for H2O2 is made, which is based on the immobilization of peroxidase in the blend membrane of RSF and PVA. Performance and characteristics of the sensor were evaluated with respect to response time, detection limit, selectivity, and dependencies on temperature and pH as well as on operating and storage stability.