An amperometric phenazine methosulphate-mediated sensor sensitive to lactate was fabricated, which was based on immobilization of lactate oxidase and horseradish peroxidase in a novel composite membrane of poly(vinyl alcohol) and regenerated silk fibroin. The water absorbability and mechanical properties of the composite membrane were investigated. Horseradish peroxidase (HRP) was employed in the catalytic reduction of hydrogen peroxide, formed by the lactate oxidase reaction, to amplify the amperometric response of the lactate sensor. In this bienzyme configuration, phenazine methosulphate was used as an electron transfer mediator between immobilized HRP and a glassy carbon electrode. Effects of pH, temperature, applied potential and concentration of phenazine methosulphate on the steady-state bioelectrocatalytic oxidation of lactate at the sensor were evaluated. Dependence of Michaelis-Menten constant K-m(app) on the concentration of phenazine methosulphate and applied potential was also investigated. The response of the sensor to lactate reached 95% steady-state current within 20 s.