Regenerated silk fibroin prepared from waste silk was used for the first time as the immobilization matrix of peroxidase. The structures of the blend membranes of regenerated silk fibroin and peroxidase were investigated using IR and scanning electron microscopy. It was found that the two macromolecules were immiscible despite their intermolecular interactions. An amperometric H2O2 sensor using phenazine methosulphate as the electron transfer agent between horseradish peroxidase in a regenerated silk fibroin membrane and glassy carbon electrode was fabricated. The sensor was highly sensitive to H2O2 with a detection limit of 1.0 X 10(-7) M H2O2 and a response time of less than 5 s. The effect of the applied potential and the mediator concentration on the Michaelis-Menten constant was calculated, and the influence of various experimental parameters such as pH, temperature, applied potential and the mediator concentration was investigated for optimum analytical performance.