Novel xanthine biosensors were successfully fabricated by immobilizing xanthine oxidase on polyvinylferrocenium perchlorate matrix (PVF+ClO4−) and platinum electrodeposited polyvinylferrocenium perchlorate matrix. PVF+ClO4− film was coated on Pt electrode at +0.7V vs. Ag/AgCl by electrooxidation of polyvinylferrocene (PVF). Platinum nanoparticles were deposited on PVF+ClO4− electrode by electrochemical deposition in 2.0mM H2PtCl6 solution at −0.2V. Xanthine oxidase was incorporated into the polymer matrix via ion exchange process by immersing modified Pt electrodes in the enzyme solution. The amperometric responses of the biosensors were measured via monitoring oxidation current of hydrogen peroxide at +0.5V. Under the optimal conditions, the linear ranges of xanthine detection were determined as 1.73×10−3–1.74mM for PVF+XO− and 0.43×10−3–2.84mM for PVF+XO−/Pt. The detection limits of xanthine were 5.20×10−4mM for PVF+XO− and 1.30×10−4mM for PVF+XO−/Pt. Moreover, the effects of applied potential, electrodeposition potential, H2PtCl6 concentration, amount of electrodeposited Pt nanoparticles, thickness of polymeric film, temperature, immobilization time, xanthine and xanthine oxidase concentrations on the response currents of the biosensors were investigated in detail. The effects of interferents, the operational and storage stabilities of biosensors and the applicabilities to drug samples of the biosensors analysis were also evaluated.