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The photophysical and biological properties of two new phenanthroline-based ligand ruthenium complexes were investigated in detail. Their DNA interaction modes were determined to be the intercalation mode using spectra titration and viscosity measurements. Under irradiation, obvious photo-reduced DNA cleavages were observed in the two complexes via singlet oxygen generation. Furthermore, complex <b>2</b> showed higher DNA affinity, photocleavage activity, and singlet oxygen quantum yields than complex <b>1</b>. The two complexes showed no toxicity towards tumor cells (HeLa, A549, and A375) in the dark. However, obvious photocytotoxicities were observed in the two complexes. Complex <b>2</b> exhibited large PIs (phototherapeutic indices) (ca. 400) towards HeLa cells. The study suggests that these complexes may act as DNA intercalators, DNA photocleavers, and photocytotoxic agents.