After the optimization of the sensor construction and the experimental variables involved in the Differential Pulse Voltammetric response of melatonin, detection limit of 3.80 × 10 −7 M, relative errors minor than 3.8% and relative standard deviation lower than 4.4% were obtained. Moreover, stoichiometry of the resulting complex has also been determined. Electrochemical Impedance Spectroscopy experiments allowed us to study the charge transfer process during melatonin oxidation. The sensing platform was topographically characterized by Atomic Force Microscopy. For the sensor construction cucurbituril suspensions were prepared in water, and MoS 2 nanosheets were obtained by liquid exfoliation in ethanol:water. We present the development of an electrochemical sensor towards melatonin determination based on the synergistic effect between MoS 2 nanosheets and cucurbituril.