Abstract A hydrogen peroxide permselective membrane with asymmetric structure was prepared and d-glucose oxidase (EC 126.96.36.199) was immobilized onto the porous layer. The activity of the immobilized d-glucose oxidase membrane was 0.34 units cm −2 and the activity yield was 6.8% of that of the native enzyme. Optimum pH, optimum temperature, pH stability and temperature stability were found to be pH 5.0, 30–40°C, pH 4.0–7.0 and below 55°C, respectively. The apparent Michaelis constant of the immobilized d-glucose oxidase membrane was 1.6 × 10 −3 mol l −1 and that of free enzyme was 4.8 × 10 −2 mol l −1. An enzyme electrode was constructed by combination of a hydrogen peroxide electrode with the immobilized d-glucose oxidase membrane. The enzyme electrode responded linearly to d-glucose over the concentration 0–1000 mg dl −1 within 10 s. When the enzyme electrode was applied to the determination of d-glucose in human serum, within day precision ( CV) was 1.29% for d-glucose concentration with a mean value of 106.8 mg dl −1. The correlation coefficient between the enzyme electrode method and the conventional colorimetric method using a free enzyme was 0.984. The immobilized d-glucose oxidase membrane was sufficiently stable to perform 1000 assays (2 to 4 weeks operation) for the determination of d-glucose in human whole blood. The dried membrane retained 77% of its initial activity after storage at 4°C for 16 months.