The complex characteristics of p-sulfonated calix arene (SC6A) and Melatonin (MLT) were examined through spectrofluorimetry, 1HNMR spectroscopy and molecular modeling calculations. The fluorescence of MLT significantly quenched upon the addition of SC6A, which revealed the formation of inclusion complexes between MLT and SC6A. The stoichiometric ratio of 1:1 was obtained via the continuous variation method. The experimental results show that SC6A forms at least 1000 times stronger inclusion complexes with MLT than âÂCD and CB, which have the similar size of the cavity. The 1HNMR spectra verified that the MLT may be partially penetrated into the hydrophobic cavity of SC6A. This finding was also confirmed by density functional theory calculations. The study is expected to provide important insight into the interactions of the physiologically important MLT with macrocyclic supramoleculars. It can also be as a fluorescence probe and sensor to detect non-fluorescent or weakly fluorescent substances.