Duplex DNA adopts a right-handed B-DNA conformation under physiological conditions. Z-DNA, meanwhile, has a left-handed helical structure and is in equilibrium with right-handed B-DNA. We recently reported that the bisnaphthyl maleimide–spermine conjugate (1) induced a B- to Z-DNA transition with high efficiency at low salt concentrations. It was also found that the bisnaphthyl ligand (1) spontaneously transformed into the corresponding [5]helicene derivative (2). Because [5]helicene 2 can potentially be chiral and because the chiral discrimination of B- and Z-DNA is also of interest, we became interested in whether enatiomerically pure [5]helicene–spermine conjugates might discriminate the chirality of B- or Z-DNA. In this study, we have demonstrated an efficient synthesis of chiral DNA-binding ligands by the conjugation of a [5]helicene unit with a spermine unit. These chiral helicene ligands exhibited recognition of B- and Z-DNA, with (P)-3 displaying preference for B-DNA and (M)-3 for Z-DNA. The characteristic features of the helicene–spermine ligands developed in this study include two points: the cationic spermine portion produces electrostatic interactions along the phosphate backbone of the minor groove, and the helicene forms complexes in an end-stacking mode. Such binding modes, together with the thermodynamic parameters, account for the mode of chiral recognition of (P)- and (M)-3 for B- and Z-DNA.