In this paper, nano-TiO2 was functionalized by different methods, and its genotoxicity and cytotoxicity were studied in detail. The genotoxicity of nano-TiO2 was evaluated by observing its interactions with pUC19 plasmid DNA at a single molecule level using atomic force microscopy. The results show that with the assistance of UVA radiation, RGDC functionalized nano-TiO2 induced less damage to plasmid DNA than unmodified ones. The HeLa cell-specific PDT effect was investigated by cytotoxicity assay correspondingly. RGDC-functionalized nano-TiO2 shows the highest killing effect to HeLa cells with the assistance of UVA radiation. The reasons that cause the contradiction between genotoxicity and cytotoxicity were analyzed, and the molecular mechanisms of the PDT effects were discussed. The results show that the genotoxicity of nano-TiO2 to plasmid DNA and its cytotoxicity to HeLa cells are related but also different. The RGDC functionalization is an effective method to increase the cytotoxicity of nano-TiO2.