In this study, xanthine molecules which can serve as an indicator of meat spoilage were determined using a novel and sensitive amperometric xanthine biosensor. Biosensor was developed by preparing a nanocomposite film that was constructed by embedding reduced expanded graphene oxide (REGO) sheets decorated with iron oxide (Fe3O4) nanoparticles into poly(glycidyl methacrylate-covinylferrocene) (P(GMA-co-VFc)) phase, and by covalent immobilization of Xanthine oxidase (XOD) on the surface of P(GMA-co-VFc)/REGO-Fe3O4 nanocomposite film. Bio-analytical optimal experimental conditions such as response time, linear range, operation and storage stability, working pH and temperature were studied. Current response of linear range was detected in the range of 2-36 mu M with a sensitivity of 0.17 mu A/M, response time of similar to 3 s, and detection limit of 0.17 mu W. The resulting bionanocomposite xanthine biosensor was subjected to fish real sample testings where 5, 8, 10, 13, 15, and 20 days-old fish samples' xanthine content was measured. The developed biosensor was found to be applicable to real samples as a very reliable fish freshness controlling technique. (C) 2015 Elsevier Ltd. All rights reserved.