2-(4-Dimethylaminophenyl)-5-fluoro-6-(morpholin-4-yl)- 1H-benzimidazole(1) has been synthesized and characterized by 1H-NMR, MS and elemental analysis. UV-Vis spectra of the aqueous solutions at different pH values reveal that compound 1 can combine three protons. Its three protonation constants are determined by spectrophotometry and calculated by non-linear least squares. The results of steady-state fluorescence measurements indicate that a special interaction occurs between compound 1 and calf thymus DNA, of which the binding constant, Kb, is (2.30 ±0.10)×l04 L/mol. Compound 1 in the concentration range of 10-8 to 1.2×l0-6 mol/L could be used for quantitative determination of DNA.