In this study, hypoxanthine biosensor based on immobilization of xanthine oxidase on polyvinylferrocenium perchlorate matrix coated Pt electrode for detection of hypoxanthine was presented. The steady-state background current of the biosensor was measured at+0.5V, which was due to the electrooxidation of enzymatically produced H2O2. The biosensor exhibited a linear range from 2.15 × 10−3mM to 1.03mM (r = 0.9963) with a detection limit of 6.5 × 10−4mM (S/N = 3) for the detection of hypoxanthine. The storage stability of the biosensor, the effect of interference by uric acid, and the applicability to fish sample of the biosensor analysis were also evaluated.