A new and simple-to-prepare hypoxanthine biosensor has been developed using xanthine oxidase (XOD) immobilised on carbon electrode surfaces. XOD was immobilised by glutaraldehyde cross-linking on carbon film (CF) electrodes and on carbon nanotube (CNT) modified CF (CNT/CF). A comparison of the performance of the two configurations was carried out by the current response using amperometry at fixed potential; the best characteristics being exhibited by XOD/CNT/CF modified electrodes. The effects of electrolyte pH and applied potential were evaluated, and a proposal is made for the enzyme mechanism of action involving competition between regeneration of flavin adenine dinucleotide and reduction of hydrogen peroxide. Under optimised conditions, the determination of hypoxanthine was carried out at -0.2 V vs. a saturated calomel electrode (SCE) with a detection limit of 0.75 μM on electrodes with CNT and at -0.3 V vs. SCE with a detection limit of 0.77 μM on electrodes without CNT. The applicability of the biosensor was verified by performing an interference study, reproducibility and stability were investigated, and hypoxanthine was successfully determined in sardine and shrimp samples.