A highly selective and stable amperometric biosensor for the determination of the hypoxanthine (Hx) molecule was designed in this study. For this purpose, the enzyme electrode was prepared by immobilizing the xanthine oxidase (XnOx) and uricase (U) enzymes to the surface obtained by electrochemical polymerization in the presence of polypyrrole-paratoluenesulfonate (PPy-pTS) on the platinum (Pt) surface. The determination limit for the Hx molecule of the prepared biosensor was determined as 5 x 10(-6) M, and the linear working range was determined as 5 x10(-6)-5 x 10(-3) M. At the end of 27 measurements, the biosensor preserved 70% of the initial amperometric response. At the end of the fourth 8 days, the enzyme electrode was observed to maintain 26% of the initial amperometric response. TheK(M)value for Pt/PPy-pTS-XnOxU enzyme electrode system prepared by immobilizing XnOxU was found to be 0.05 mM, andV(max)was 0.56 mu A/min. The effects of the interventions in biological environments on the biosensor response were examined. Also, since this biosensor has the potential to be used for the determination of Hx in synthetic samples, it can find an important field of study in the biological and food industry.