A practical glucose biosensor was developed by combining the intrinsic peroxidase-like activity of Fe(3)O(4) nanoparticles (Fe(3)O(4) NPs) and the anti-interference ability of the nafion film. Glucose oxidase (GOD) was simply mixed with Fe(3)O(4) NPs and cross-linked on the Pt electrode with chitosan (Cs) medium by glutaraldehyde, and then covered with a thin nafion film. The biosensor showed high sensitivity (11.54 microAcm(-2)mM(-1)), low detection limit (6 x10(-6)M), and good storage stability. A linear calibration plot was obtained in the wide concentration range from 6 x10(-6) to 2.2 x10(-3)M. The modified electrode could virtually eliminate the interference during the detection of glucose. Furthermore, the biosensor was successfully applied to detect glucose in human serum sample. This fabrication of glucose biosensor was of considerable interest due to its promise for simple procedure and optimizing conditions in practical application.