In this study, water-soluble BODIPY dyes (BDPY-4, BDPY-5) bearing dimethylamino and diethylamino groups were synthesized for the first time. The DNA interaction and nuclease effect of two water-soluble BODIPY dyes were examined using absorption, competitive binding experiments, and agarose gel electrophoresis. Then, cytotoxicity and phototoxicity of the compounds were determined against A549 cells by MTT cell viability assay. The ultraviolet-visible (UV-vis) spectra of BDPY-4 and BDPY-5 displayed hypochromism and absorption maxima shifted to the red region. In addition, K-b values of the compounds were calculated as 2.23 x 10(4) and 4.84 x 10(4) M-1, respectively. An high-performance liquid chromatography (HPLC) instrument without a column was used to conduct competitive binding experiments for the first time. The approach prevented the decrease in the response due to dilution and enabled the analysis to be carried out in a short time. Upon increasing concentrations of the compounds, the emission intensity of EB-DNA significantly decreased. These obtained results suggested that the compounds bound to CT-DNA with intercalative mode. At the same time, the Stern-Volmer constants were determined as 1.80 x 10(6) and 1.79 x 10(6) M-1 for BDPY-4 and BDPY-5, respectively, which is coherent with intercalative binding mode. The compounds did not display noticeable photonuclease actions with light irradiation. BDPY-4 and BDPY-5 showed moderate phototoxic properties on light irradiation.