Complexation between Rhodamine B (RB) and sulfonated calix [n] arenes ( SCnA) were studied by means of UV-vis spectroscopy and fluorescence spectroscopy. In the presence of sulfonated calix [n] arenes, the absorption band of Rhodamine B shifts to longer wavelength and its intensity decreases. The formation of a host-guest type complex also results in the fluorescence quenching of Rhodamine B. The association constants for the RB/SCnA complexes increase in the order of SC4A < SC6A < SC8A and show dependence on the size of the cavities of the calixarenes. The fluorescence is selectively regenerated by adding acetylcholine. Based on this observation, a method to determine acetylcholine in a real neutral aqueous medium was developed.