In this work a novel electrochemical biosensing platform based on the coupling of two different nanostructured materials (gold nanoparticles and fullerenols) displaying interesting electrochemical features, has been developed and characterized. Gold nanoparticles (AuNPs) exhibit attractive electrocatalytic behavior stimulating in the last years, several sensing applications; on the other hand, fullerene and its derivatives are a very promising family of electroactive compounds although they have not yet been fully employed in biosensing. The methodology proposed in this work was finalized to the setup of a laccase biosensor based on a multilayer material consisting in AuNPs, fullerenols and Trametes versicolor Laccase (TvL) assembled layer by layer onto a gold (Au) electrode surface. The influence of different modification step procedures on the electroanalytical performance of biosensors has been evaluated. Cyclic voltammetry, chronoamperometry, surface plasmon resonance (SPR) and scanning tunneling microscopy (STM) were used to characterize the modification of surface and to investigate the bioelectrocatalytic biosensor response. This biosensor showed fast amperometric response to gallic acid, which is usually considered a standard for polyphenols analysis of wines, with a linear range 0.03–0.30mmolL−1 (r2=0.9998), with a LOD of 0.006mmolL−1 or expressed as polyphenol index 5.0–50mgL−1 and LOD 1.1mgL−1. A tentative application of the developed nanostructured enzyme-based biosensor was performed evaluating the detection of polyphenols either in buffer solution or in real wine samples. • An electrochemical biosensing platform based on the coupling of two different nanostructured materials (gold nanoparticles and fullerenols) was developed. • The modification procedure has been checked by Surface Plasmon Resonance (SPR) and the obtained surface was characterized by Scanning Tunneling Microscopy (STM). • The electrochemical characterization in presence of redox mediators indicates that the nanomaterials improve the electron-transfer kinetic of the processes. • The laccase enzyme from Trametes versicolor, once immobilized on the electrodic surface, retains catalytic activity and displays an increase of bioelectrochemical performances. • The obtained laccase biosensor displays good stability and sensitivity when operating in flow injection analysis towards real wine samples.