Two biotin-appended dansylmodified cyclodextrins 3 and 4 were prepared to examine the effects of avidin binding on guest-inclusion phenomena and fluorescence properties of cyclodextrin derivatives. The fluorescence intensities of hosts 3 and 4 are more than three times larger in the presence of avidin than those in the absence of avidin. The fluorescence lifetime measurements indicate that the dansyl moiety is not exposed to bulk water in the presence of avidin. The longer lifetime component, which newly appears in the presence of avidin, is likely to be that of the dansyl moiety located in the hydrophobic region of avidin. The addition of bile acids such as hyodeoxycholic acid resulted in decreases in the fluorescence intensities of 3 and 4 in accordance to the extrusion of the dansyl moiety from the hydrophobic cyclodextrin cavity to bulk water. Similar guest-induced decreases in the fluorescence intensity were observed for 3 in the presence of avidin, but under the same conditions the decreases are smaller for 4. On the other hand, the binding constants of 3 and 4 became larger in the presence of avidin. All these results demonstrate that avidin perturbs the inclusion phenomena and fluorescence properties of 3 and 4 by providing a hydrophobic environment around the host cavity.