The preparation and performance of xanthine oxidase electrochemical biosensor developed for the determination of hypoxanthine is reported. Xanthine oxidase (XOD) and a mediator potassium ferrocyanide (K4Fe(CN) were entrapped into a polypyrrole (PPy) film during galvanostatic film formation. The optimum conditions for the formation of PPy-XOD-Fe(CN)6 4a?? film include 0.4M pyrrole, 6.2 U/mL XOD, 40mMK4Fe(CN)6, polymerisation period of 200 s, and an applied current density of 0.5mA/cm2. Two modes of detection, the potentiometric and amperometric modes, were investigated. The optimum potential for the amperometric biosensing of hypoxanthine was 800mV vs. Ag/AgCl (3M KCl) in 0.05M phosphate buffer. A comparison of the sensitivities for the amperometric and potentiometric modes revealed that potentiometric detection was more sensitive and amperometric detection gave a wider linear concentration range. The PPy-XOD biosensor was successfully used for the determination of fish freshness.