Stable and transient spectroscopic studies were conducted to investigate the influence of DNA phosphate bases in the process of DNA damage photo-induced by ciprofloxacin (CPX). The results of UV-visible and fluorescence studies confirmed that the absorption and emission properties of CPX were affected by the concentration of phosphate buffer (PB), and these results indicated that there are interactions between CPX and the phosphate anion. To investigate the relationship between the phosphate base and CPX, a comparative experiment was conducted using guanosine (Gua), 2'-deoxyguanosine (dG), and 2'-deoxyguanosine-5'-monophosphate (dGMP). By comparative experiments, we found that the type of spectrum of CPX triplet state was changed due to the phosphate base of dGMP, hence, we concluded that the photo-damage process was changed. The effects of phosphate base of dGMP on the CPX triplet state ((CPX)-C-3*) spectrum have been investigated by laser flash photolysis methods, and the results confirmed that there are hydrogen-bonding interactions between the phosphate base of dGMP and CPX. In this study, we found that CPX and dGMP are combined together by the hydrogen bond which changed the mode of DNA damage photo-induced by CPX. Finally, based on the results obtained in this study, a rational scheme to describe the role of phosphate base playing in the DNA damage process photo-induced by CPX is proposed.