An electrochemical biosensor based on manganese oxide (Mn3O4) and chitosan (Cn) nanocomposite has been fabricated for fish freshness detection. The electrophoretic deposition of Mn3O4 nanoparticles (15–20 nm) with Cn has changed their morphological arrangement leading to pearl shaped of Mn3O4–Cn nanocomposite on indium tin oxide substrate. Size and morphology of nanocomposite have been confirmed by high resolution transmission electron microscopy (HRTEM), X-ray diffraction (XRD) and scanning electron microscopy (SEM). The results of electrochemical response reveal that this improved sensor has widest detection range of xanthine concentration from 1 to 500 µM and excellent sensitivity of 1.46 μA µM−1 cm−2. The fabricated XOx/Mn3O4–Cn/ITO biosensor can detect as low as 1.31 μM of xanthine and lower Km value of 0.018 μM confirming its superior affinity towards the nanocomposite film.