A glucose biosensor using a glucose oxidase (GO(x))-immobilized nylon net with glutaraldehyde as cross-linking reagent and an oxygen (O-2) electrode for the determination of glucose has been fabricated. The detection scheme was based on the utilization of dissolved O-2 in oxidation of glucose by the membrane bound GO(x). Crucial factors including O-alkylation temperature, reaction times of nylon net with dimethyl Sulfate, L-lysine, and glutaraldehyde, and enzyme loading were examined to determine the optimal enzyme immobilization conditions for the best sensitivity of the developed glucose biosensor. In addition, the effects of pH and concentration of phosphate buffer on the response of the biosensor were Studied. The glucose biosensor had a linear range of 18 mu M to 1.10 mM with the detection limit of 9.0 mu M (S/N = 3) and response time of 80s. The biosensor exhibited both good operational stability with over 200 measurements and long-term storage stability. The results from this biosensor compared well with those of a commercial glucose assay kit in analyzing human serum glucose samples. (C) 2008 Elsevier Inc. All rights reserved