An electrochemical biosensor based on xanthine oxidase (XOx), titanium dioxide nanoparticles and carboxylated multi-walled carbon nanotubes (TiO2/c-MWCNT) nano-composites for sensitive detection of xanthine has been developed. TiO2/MWCNT nano-composites were used as the sensing platform in order to immobilize XOx and magnify the sensor response. FTO electrode was employed to amplify electrochemical signal in the buffer solution. Detailed morphological, electrochemical, structural and optical characterization of XOx/TiO2-NPs/c-MWCNT/FTO electrode was done using XRD, DLS, SEM, EIS, CV and shows quick response time (within 30s), linearity as 0.5- 500 µM, lower detection value of 0.05 micromolar with signal: noise ratio of 3, excellent reproducibility, high selectivity and shelf life of about 8 weeks under refrigerated conditions. The developed biosensor was further used to determine the xanthine levels in the labeo fish samples obtained from market. The accuracy of the developed biosensor was cross-checked by the customary enzymic colorimetric method (99% correlation). Thus, the existing research confirms the development of a highly sensitive, stable and a reliable bio-sensing method to detect the freshness of fish samples.