A glucose biosensor, which was based on self-assembled Prussian Blue (PB) modified electrode with glucose oxidase (GOD) immobilized in cross-linked glutaraldehyde matrix, was developed. Fourier-transform infrared spectroscopy shows that the immobilized GOD retains its native conformation. Cyclic voltammetry was used to examine the electrocatalytic property of the enzyme electrode. The prepared glucose biosensor exhibits fast response (<4 s) and low detection limit of 5 x 10(-6) M. The calculated apparent Michaelis constant K-M was 6.3 +/-1.2 mM, indicating a high affinity between the GOD and glucose. The effects of glutaraldehyde concentration and GOD loading on the sensitivity of the glucose biosensor have also been investigated. Under the optimal conditions, the biosensor shows a high sensitivity of about 80 mA M-1 cm(-2) in a concentration range up to 1 x 10(-3) M. The relative standard deviation (RSD) for intra-electrode and inter-electrode were 4% and 5%, respectively. In addition, the anti-interferent ability and stability of the biosensor were also discussed. (C) 2009 Elsevier Ltd. All rights reserved.