A novel composite membrane of poly(vinyl alcohol) and regenerated silk fibroin was employed to immobilize horseradish peroxidase (HRP) and IR was used to give a useful insight into the structure of the composite membrane before and after ethanol treatment. A methylene green-mediated sensor sensitive to hydrogen peroxide was fabricated, based on the composite membrane as immobilization matrix for HRP. Cyclic voltammetry and amperometric measurement were for the first time utilized to demonstrate the suitability of methylene green as an electron transfer mediator between immobilized HRP and a glassy carbon electrode in bioelectrocatalytic reduction of hydrogen peroxide. Performance and characteristics of the sensor were evaluated in respect to response time, detection limit, applied potential and concentration of the mediator. The sensor possesses a variety of characteristics including good sensitivity, rapid response time and low detection of limit of 0.1 umol/L. A novel composite membrane of poly (vinyl alcohol) and regenerated silk fibroin was employed to immobilize horseradish peroxidase (HRP) and IR was used to give a useful insight into the structure of the composite membrane before and after ethanol treatment. A methylene green-mediated sensor sensitive to hydrogen peroxide was fabricated, based on the composite membrane as immobilization matrix for HRP. Cyclic voltammetry and amperometric measurement were for the first time utilized to demonstrate the suitability of methylene green as an electron transfer mediator between immobilized HRP and a glassy carbon electrode in bioelectrocatalytic reduction of hydrogen peroxide. Performance and characteristics of the sensor were evaluated in respect to response time, detection limit, applied potential and concentration of the mediator. The sensor possesses a variety of characteristics including good sensitivity, rapid response time and low detection of limit of 0.1 umol / L.