A new nanocomposite based on gold nanoparticles supported on mercaptoethanol bonded to melamine modified multi-walled carbon nanotubes (MWCNTs/CC-SH/AuNPs) was synthesized, characterized and used as sensitive sensor for the determination of levothyroxine (T4) using glassy carbon electrode. In order to characterize the nanocomposite, different methods including field emission scanning electronic microscopy (FE-SEM), Fourier transform infrared (FTIR), transmission electron microscopy (TEM), energy dispersion X-ray spectroscopy (EDX) and Raman spectroscopy were used. The differential pulse voltammetry data indicated that the acquired response was linearly reliant on the T4 concentration within 10-120 nM, with a detection limit of 2.84 nM.