Binding behavior of L- and D-tryptophan-modified β-cyclodextrins (L/D-Trp-β-CD) (1 and 2) with four bile acids, i.e., cholate (CA), deoxycholate (DCA), glycocholate (GCA), and taurocholate (TCA), has been investigated by fluorescence, circular dichroism, and 2D-NMR spectroscopies and fluorescence lifetime measurement, as well as isothermal titration microcalorimetry. From the induced circular dichroism (ICD) and 2D NMR spectra, it is deduced that the D-Trp moiety of 2 attached to β-CD is more deeply self-included in the cavity than that of the antipodal L-Trp moiety of 1, indicating appreciably enantioselective binding of the chiral sidearm by β-CD. Interestingly, the original difference in conformation between 1 and 2 led to quite a large difference in affinity toward DCA, giving 3.3 times higher binding ability for 2 than for 1. Thermodynamically, the inclusion complexation of 1 and 2 with bile acids was entirely driven by favorable enthalpy change (ΔH°) with accompanying negative entropy change (ΔS°). The stronger binding of bile acids by L/D-Trp-β-CD is attributable to higher enthalpic gains. The combined use of the calorimetric and NMR ROESY spectral examinations revealed the correlation between the thermodynamic parameters and the role of sidearm conformation in modified β-cyclodextrins.