Xanthine oxidase (XO) was successfully immobilized by covalent attachment on poly(glycidyl methacrylate-co-vinylferrocene) (P(GMA-co-VFc)), a redox copolymer containing pendant epoxy and ferrocene moieties, for the evaluation of both the biosensing properties and the effect of the interaction of ketoconazole (Ktc) with the immobilized XO. The binding interaction between Ktc, a drug used to treat fungal infections, and the immobilized XO on P(GMA-co-VFc) was also studied by fluorescence spectroscopy technique. The binding capacity of the drug was determined using a calibration curve equation that was drawn at excitation wavelength of 300 nm using fluorescence spectroscopy. The interaction ability was confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and native-PAGE analysis. The enzyme electrode exhibited a linear range from 2.7 × 10−3 to 0.55 mM with a sensitivity of 19.42 μA mM−1 cm−2 and a detection limit of 8 × 10−4 mM for the detection of xanthine. The activation energy (Ea) and the apparent Michaelis–Menten constant (Kmapp) values were found to be 12.30 kJ mol−1 and 0.38 mM, respectively.