The interactions between L-tryptophan(L-Trp) and water-soluble p-sulfonatocalix[4]-arene (SCX4) were investigated by spectrofluorometry. The effect of pH was discussed. The interaction process was estimated from the thermodynamic viewpoint. The SCX4 was found to be able to adjust its conformation to fit the size of L-Trp, and the benzene of L-Trp penetrated into the hydrophobic cavity of SCX4. Meanwhile, the binding mechanism was further elucidated by 1HNMR and molecular mechanics calculations.