Flexible regioselectively hetero-substituted hosts, 6A-O-4-pyrenylbutanoyl-6X-O-p-cyanobenzoyl-modified γ-cyclodextrins (X = B or H, C or G, D or F, and E for γ-1, γ-2, γ-3, and γ-4, respectively) have been synthesized in order to investigate their fluorescence sensing properties for application to organic compounds such as bile acids and cyclic alcohols. The hosts, γ-1, γ-2 and γ-3, exhibit both monomer and exciplex fluorescence, whereas γ-4 exhibits only monomer emission. In guest-induced emission of γ-1, γ-2 and γ-3, exciplex emission is suppressed while monomer emission is increased. However, γ-4 exhibits only a negligible change in monomer fluorescence in the presence of guests. The extent of exciplex fluorescence variation of γ-1, γ-2 and γ-3 with guests is recognized as the manifestation of the sensing ability of the hosts. A sensing parameter (ΔIex/I0ex) was used to describe the sensing ability of three hosts. Host γ-analogs, γ-1, γ-2 and γ-3, are able to detect ursodeoxycholic acid, deoxycholic acid, chenodeoxycholic acid, and (−)-borneol with high sensitivity by exciplex emission. The sequence of the binding ability of these hosts is γ-2 > γ-1 > γ-3. The behaviors of the appended moieties of these hosts during the formation of host–guest complexes were studied using induced circular dichroism (ICD) and fluorescence spectra. The host γ-analogs γ-1–γ-3 exhibit different ICD patterns to γ-4 before and after addition of ursodeoxycholic acid. The guest-induced variations of ICD and fluorescence spectra changes suggest that the pyrene and cyanobenzene moieties move, altering the spatial relationship between them.