In this work, the interactions between ethidium bromide (ET) and water-soluble sulfonate calix[n]arenes (n: 4, 6, and 8) were investigated by NMR, FT-IR, and fluorescence spectroscopic methods. The aim was to evaluate both the stoichiometry and the mechanism of the possible complex structure between sulfonate calix[n]arenes and ET. The spectroscopic data revealed that a 1:1 binding mode between calixarene and ET was occurred. Furthermore, thermodynamic parameters and fluorescence titration experiments were studied at different temperatures to determine both the quenching mechanism and the type of intermolecular forces in complex formation. Host-guest complexation of sulfonate calix[n]arenes and ET could be used to overcome some adverse effects related to the using of ethidium bromide during biological applications as a DNA marker treatment.