The first member of the single-isomer, dicationic cyclodextrin (CD) family, 6A-ammonium-6C-butylimidazolium--cyclodextrin chlorides (AMBIMCD), has been synthesized, analytically characterized, and used to separate a variety of acidic enantiomers and amino acids by CE. Starting from mono-6A-azido--cyclodextrin, the cationic imidazolium and ammonium moieties were subsequently introduced onto primary ring of -cyclodextrin via nucleophilic addition and Staudinger reaction. The analytically pure AC regio-isomer CD was further obtained via column chromatography. This dicationic CD exhibited excellent enantioselectivities for selected analytes at concentration as low as 0.5 mM, which were even better than those of its mono-imidazolium or ammonium-substitued counterpart CDs at 10 equivalent concentrations. The effective mobilities of all studied analytes were found to decrease with the concentration of AMBIMCD. Inclusion complexation in combination with eletrostatic interactions seemed to account for the enhanced chiral discrimination process.