A novel nanobiocomposite for immobilization of xanthine oxidase (XO) was developed by incorporating functionalized MWCNT in nanogold doped poly(o-phenylenediamine) (PPD) (Au–PPD) film on glassy carbon electrode (GCE) for selective and sensitive detection of xanthine in real samples e.g. blood, urine, fish. Stable colloid of o-phenylenediamine (OPD) and HAuCl₄ in acidic environment was electropolymerized on working electrode (GCE) to form an ultrathin film of AuNP–PPD which possessed permselectivity and no interference against electroactive species such as ascorbic acid and uric acid. Spectrophotometric and microscopic analysis confirmed the doping behaviour of AuNP. Electrodeposition of carboxylated MWCNT onto the Au–PPD film increased conductivity, sensitivity and also facilitated a microenvironment to entrap XO enzyme by covalent bonding, enhancing storage stability. The conductive nature of the electrode after every step of modification was investigated by electrochemical impedance spectroscopy. High I_max/K_map value was achieved by the XO/fMWCNT/Au–PPD modified electrode. Oxidation of xanthine on this modified electrode was diffusion-controlled involving two electrons in the rate-determining step with a transfer coefficient (α) of about 0.596. Differential pulse voltammetric study of XO/fMWCNT/Au–PPD/GCE exhibited good analytical characteristics e.g. low detection limit (12 nM) (S/N = 3), a wide linear range of 0.01–300 μM (R² = 0.994), good sensitivity (14.03 μA μM⁻¹ cm⁻²), fast response (6 s) at anodic potential of +0.625 V vs. Ag/AgCl (pH 7.0). It retained 91% of its initial activity even after 210 times of use over a period of 4 months when stored at 4 °C. The applicability of the xanthine biosensor was tested by performing reproducibility, repeatability and interference study on real samples.