An xanthene dye, pyronine Y, was found to exhibit opposite fluorescence behaviors at different pH values in the presence of N,N’-bis(2- aminoethyl)-2,2’-biquinoline-4,4’-dicarboxamide-bridged bis(β-cyclodextrin)(2). That is, pyronine Y showed the quenched fluorescence in an acidic(pH 2.0) or a neutral(pH 7.2) environment, while the enhanced fluorescence in a basic environment(pH 12.0), with the addition of 2. Further studies by fluorescence titrations and 2D NMR indicated that different binding modes of positively charged and neutral PY molecules upon complexation with bis(β-cyclodextrin) 2 should be responsible for opposite fluorescence behaviors. This result may enable the biquinolino-bridged bis(β-cyclodextrin) as an efficient chemical sensor for the protonation and deprotonation of xanthene dyes.