Spectrofluorimetric titrations were performed in aqueous phosphate buffer (pH 7.20, 0.1 mol dm−3) to determine the binding constants of β‐cyclodextrin (1), mono(6‐O‐α‐maltosyl)‐β‐cyclodextrin (2) and mono[2‐O‐(2‐hydroxypropyl)]‐β‐cyclodextrin (3) with four fluorescent dyes, ammonium 8‐anilino‐1‐naphthalenesulfonate (ANS), sodium 2‐(p‐toluidino)naphthalene‐6‐sulfonate (TNS), Acridine Red (AR) and Rhodamine B (RhB). The fluorescence of ANS, TNS and AR were enhanced, whereas that of RhB was quenched, by the inclusion complexation with β‐cyclodextrin hosts 1–3. It was found that the binding ability of the three cyclodextrin hosts generally decreases in the order 1 > 3 > 2, which indicates that the hydrophobicity of the substituent affects the original binding ability of parent β‐cyclodextrin to some extent. On the other hand, the size, shape and charge of the guest are the crucial factors which dominate the stability of the supramolecular complex formed. Copyright © 2000 John Wiley & Sons, Ltd.