We present the development of an electrochemical sensor towards melatonin determination based on the synergistic effect between MoS₂ nanosheets and cucurbit[8]uril. For the sensor construction cucurbit[8]uril suspensions were prepared in water, and MoS₂ nanosheets were obtained by liquid exfoliation in ethanol:water. The sensing platform was topographically characterized by Atomic Force Microscopy. Electrochemical Impedance Spectroscopy experiments allowed us to study the charge transfer process during melatonin oxidation. Moreover, stoichiometry of the resulting complex has also been determined. 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. The proposed sensor has been successfully applied to melatonin determination in pharmaceutical and biological samples as human urine and serum, with very good recoveries ranging from 90 to 102%.