Biosensor highly sensitive to hydrogen peroxide has constructed by immobilizing horseradish peroxidase in Eastman-AQ-N-methyl phenazine methosulphate modified electrode via cross-linking. Cyclic voltammetry and chronamperometry were employed to demonstrate the effective electron transfer between immobilized horseradish peroxidase and a glassy carbon electrode via N-methyl phenazine methosulphate in Eastman-AQ polymer film. Because of high efficiency of bioelectrocatalytic reduction of hydrogen peroxide via N-methyl phenazine methosulphate, the hydrogen peroxide sensor was combined with glucose oxidase and β-galactosidase for bienzyme and trienzyme-based biosensor for determination of low glucose and lactose. The biosensors for hydrogen peroxide, glucose and lactose possessed a wide variety of advantages including long stability, rapid response times, wide dynamic range, high sensitivity and selectivity. Comparison of glucose biosensor with colorimetric method with glucose oxidase and peroxidase for the determination of serum glucose from diabetic patients indicates that the results display a good consistency.