A biosensor is developed for the determination of hypoxanthine (Hx) by entrapment of XOD and a mediator, ferrocene or potassium ferrocyanide, (K4Fe(CN)6, into a PPy film during galvanostatic film formation. The optimum conditions for the formation of PPy-XOD-Fe(CN)64- film include 0.4 M pyrrole, 6.2 U mL-1 XOD, 40 mM K4Fe(CN)6, polymerisation period of 200 sec and an applied current density of 0.5 mA cm-2. Potentiometric modes of detection, was investigated. The potential for the potentiometric biosensing of Hx was 0.0 mV vs. Ag/AgCI (3 M KCl) in 0.05 M phosphate buffer. It is revealed that potentiometric detection was sensitive and gave a wider linear concentration range. The PPy-XOD potentiometric biosensor was successfully used for the determination of fish freshness.