A new amperometic biosensor for hydrogen peroxide based on methylene blue incorporated into Nafion membrane as electron transfer mediator was fabricated. It was found that methylene blue incorporated into Nafion membrane by ion-exchanging could effectively transfer electrons between horseradish peroxidase and glassy carbon electrode. Bio-electrocatalytic reduction of hydrogen peroxide at the biosensor was evaluated with respect to solution pH, temperature, operating potential and influences of ascorbic acid etc. The biosensor response exhibited fine selectivity, high sensitivity and a linear dependence on the analytic concentration range 5×10^－7～2×10^－4 mol/L. Response time was less than 30 s.