The nanoparticles of xanthine oxidase (XODNPs) were prepared by desolvation method and characterized by transmission electron microscopy(TEM) and Fourier transform Infra-red spectroscopy(FTIR). These XODNPs were immobilized directly onto surface of Au electrode via covalent bond. An improved amperometric xanthine biosensor was fabricated using XODNPs modified Au electrode, as working electrode (WE), Ag/AgCl as reference electrode and Pt wire as auxiliary electrode connected through potentiostat.The biosensor showed optimum current at 0.25 V within 5 s, at pH 7.0 and 35 °C.The working range of biosensor was 0.01–1.0 μM with a limit of detection of 0.01 μM.The % recovery of added xanthine (1 and 5 μM) was 94.1 ± 0.2 and 94.8 ± 0.31 respectively. Within and between batch coefficient of variation (CV) were <2% & < 2.5% respectively. A good correlation (R2 = 0.996) was obtained between xanthine contents in various fish meat,as measured by the present biosensor and the standard bienzymic colorimetric method. The biosensor was employed for measuring xanthine level in fish meat at different stages of storage to test its freshness. The XODNPs modified Au electrode was stored dry at 4 °C for 2 months.