A reagentless biosensor highly sensitive to hydrogen peroxide was constructed by immobilizing horseradish peroxidase on Nafion(R)-new methylene blue N modified electrode. Cyclic voltammetry and chronamperometry were for the first time employed to demonstrate the feasibility of electron transfer between immobilized horseradish peroxidase and a glassy carbon electrode via new methylene blue N incorporated in Nafion(R) gel. Performance and characteristics of the sensor were evaluated with respect to response time, detection limit, selectivity, and dependence on applied potential, thickness of Nafion(R) membrane, ionic strength, temperature and pH as well as operating and storage stability. High sensitivity of the sensor with a detection limit of 0.5 mu M was due to high efficiency of the electron communication between immobilized horseradish peroxidase and the electrode via new methylene blue N.