AbstractFunctionalized graphene-based nanocomposites have opened new windows to address some challenges for increasing the sensitivity, accuracy and functionality of biosensors. Polyaniline (PANI) is one of the most potentially promising and technologically important conducting polymers, which brings together the electrical features of metals with intriguing properties of plastics including facile processing and controllable chemical and physical properties. PANI/graphene nanocomposites have attracted intense interest in various fields due to unique physicochemical properties including high conductivity, facile preparation and intriguing redox behavior. In this article, a functionalized graphene-grafted nanostructured PANI nanocomposite was applied for determining the ascorbic acid (AA) level. A significant current response was observed after treating the electrode surface with methacrylated graphene oxide (MeGO)/PANI nanocomposite. The amperometric responses showed a robust linear range of 8–5,000 µM and detection limit of 2 µM (N = 5). Excellent sensor selectivity was demonstrated in the presence of electroactive components interfering species, commonly found in real serum samples. This sensor is a promising candidate for rapid and selective determination of AA.