The screen-printed three-electrode system was applied to fabricate a new type of disposable amperometric xanthine oxidase biosensor. Carbon-working, carbon-counter and Ag/AgCl reference electrodes were all manually printed on the polyethylene terephthalate substrate forming the screen-printed three-electrode system by the conventional screen-printing process. As a mediator, Prussian blue could not only catalyze the electrochemical reduction of hydrogen peroxide produced from the enzyme reaction, but also keep the favorable potential around 0 V. The optimum operational conditions, including pH, potential and temperature, were investigated. The sensitivities of xanthine and hypoxanthine detections were 13.83 mA/M and 25.56 mA/M, respectively. A linear relationship was obtained in the concentration range between 0.10 M and 4.98 M for xanthine and between 0.50 M and 3.98 M for hypoxanthine. The small Michaelis-menten constant value of the xanthine oxidase biosensor was calculated to be 3.90 M. The results indicate that the fabricated xanthine oxidase biosensor is effective and sensitive for the detection of xanthine and hypoxanthine. xanthine oxidase, xanthine, hypoxanthine, screen-printed three-electrode system, Prussian blue

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