Two new polypyridyl ligands containing substituent Br at different positions in the phenyl ring, PBIP [PBIP=2-(4-bromophenyl)imidazo[4,5-f]1,10-phenanthroline], OBIP [OBIP=2-(2-bromophenyl)imidazo[4,5-f]1,10-phenanthroline] and their Ru(II) complexes, [Ru(phen)2PBIP]2+ 1, [Ru(phen)2OBIP]2+ 2 (phen=1,10-phenanthroline), have been synthesized and characterized. The binding strength of the two complexes to calf thymus DNA (CT DNA) was investigated with spectrophotometric methods, viscosity measurements, as well as equilibrium dialysis and circular dichroism spectroscopy. The theoretical calculations for these two complexes were also carried out applying the density functional theory (DFT) method. The experimental results show that the Br group substituting H at different positions of the phenyl ring in the intercalated ligand has significant effects on the spectral properties and the DNA-binding behaviors of Ru(II) complexes. Both the complexes can bind to CT DNA in intercalative mode and interact with CT DNA enantioselectively. Moreover, complex 1 can bind to CT DNA more strongly than complex 2, and complex 2 can become a much better candidate as an enantioselective binder to CT DNA than complex 1. The theoretical calculations show that both intercalative ligands, PBIP and OBIP, in these two complexes are essentially planar, and the obtained electronic structures of the complexes can be used to explain reasonably some of their experimental regularities or trends. Such experimental and theoretical information will be useful in design of novel probes of nucleic acid structures.