This article reports a novel electrochemical sensor based on a nanocomposite for the sensitive determination of Thyroxine (T4), the active form of the hormone. Hydrodynamic amperometry is performed with a nanocomposite electrode based on the dispersion of a graphene–based filler hybrid-nanomaterial throughout an insulating epoxy resin in the optimum composition ratio (the near–percolation composition). This hybrid-nanomaterial consists of reduced graphene oxide tuned with gold nanoparticles and a biorecognition agent, the thiolated ß-cyclodextrin. Recognition of T4 is accomplished via supramolecular chemistry, due to the formation of an inclusion complex between ß-cyclodextrin and T4. The amperometric device operates at +0.85 V vs. Ag/AgCl, where the oxidation of T4 takes place on the electrode surface. The sensor covers the 1.00 nM to 14 nM T4concentration range in a 0.1 M HCl solution, with a detection limit of 1.00 ± 0.02 nM. The sensor can be easily reset by polishing. It exhibits the lowest detection limit regarding to any other electrochemical electrodes for T4 determination previously described in literature