Two beta-cyclodextrin derivatives with chiral chromophoric substituents, that is, L- (1) and D-tyrosine-modified beta-cyclodextrin (2), were synthesized and fully characterized. Their inclusion modes, binding abilities, and molecular selectivities with four steroid guests, that is, cholic acid sodium salt (CA), deoxycholic acid sodium salt (DCA), glycochoic acid sodium salt (GCA), and taurocholic acid sodium salt (TCA), were investigated by the circular dichroism, 2D NMR, and isothermal titration microcalorimetry (ITC). The results obtained from the circular dichroism and 2D NMR showed that two hosts adopted the different binding geometry, and these differences subsequently resulted in the significant differences of molecular binding abilities and selectivities. As compared with native beta-cyclodextrin and tryptophan-modified beta-cyclodextrin, host 2 showed the enhanced binding abilities for CA and DCA but the decreased binding abilities for GCA and TCA, however, host 1 showed the decreased binding abilities for all four bile salts. The best guest selectivity and the best host selectivity were K-S(2-DCA)/K-S(2-TCA) = 12.6 and K-S(2-CA)/K-S(1-CA) = 10, respectively, both exhibiting great enhancement as compared with the corresponding values of the previously reported L- and D-tryptophan-modified beta-cyclodextrins. Thermodynamically, it was the favorable enthalpic gain that led to the high guest selectivity and host selectivity.