A new ligand 3-(1H-imidazo[4,5-f][1,10]phenanthrolin-2yl)phenylboronic acid and its (IPPBA) three ruthenium(II) complexes [Ru(phen)2(IPPBA)](ClO4)2 (1), [Ru(bpy)2(IPPBA)](ClO4)2 (2) and [Ru(dmb)2(IPPBA)](ClO4)2 (3) have been synthesized and characterized by elemental analysis, UV/VIS, IR, 1H-NMR,13C-NMR and mass spectra. The binding behaviors of the three complexes to calf thymus DNA were investigated by absorption spectra, emission spectroscopy, viscosity measurements, thermal denaturation and photoactivated cleavage. The DNA-binding constants for complexes 1, 2 and 3 have been determined to be 7.9 × 105 M−1, 6.7 × 105 M−1 and 2.9 × 105 M−1. The results suggest that these complexes bound to double-stranded DNA in an intercalation mode. Upon irradiation at 365 nm, three ruthenium complexes were found to promote the cleavage of plasmid pBR322 DNA from super coiled form І to nicked form ІІ. Further in the presence of Co2+, the emission of DNA–Ru(ΙΙ) complexes can be quenched. And when EDTA was added, the emission was recovered. The experimental results show that all three complexes exhibited the “on–off–on” properties of molecular “light switch”. The highest Cytotoxicity potential of the complex1 was observed on the Human alveolar adenocarcinoma (A549) cell line. Good agreement was generally found between the spectroscopic techniques and molecular docked model which provides further evidence of groove binding.