Glassy carbon paste electrode based on xanthine oxidase (XO) was developed for the determination of xanthine (X). The biosensor employs the amperometric detection of hydrogen peroxide evaluated by the enzymatic reaction catalyzed by XO immobilizing in the glassy carbon paste material. The optimum operational conditions for the biosensor were investigated and the system was calibrated for both hypoxanthine and xanthine, respectively. A linearity for the biosensor was obtained in concentration range between 5.0 × 10−7– 4.0 × 10−5 M xanthine and 2.0 × 10−5–8.0 × 10−5 M hypoxanthine with detection limits of 1.0 × 10−7 and 5.3 × 10−6 M, respectively. Furthermore, as well as optimization of enzyme amount, sample application and accuracy were also searched.