The microenvironmental properties as well as the chiral discrimination abilities of four kinds of bile salt micelles were investigated by the fluorescence probe technique. A new fluorescence probe, 1,1'-bi-2-naphthol, was exploited to study the aggregation of bile salts, size, micropolarity, and microrigidity of the micelles with or without the presence of inorganic salts. Based on these results, the chiral discrimination abilities of bile salt micelles were further investigated by using (R)- and (S)-1,1'-bi-2-naphthol as chiral fluorescence probes. Different chiral discrimination ability was revealed by fluorescence spectra, fluorescence increase rate, and fluorescence quenching constants. The chiral discrimination mechanism of bile salt micelles was discussed.