Abstract: AimTo study the binding mode of balofloxacin with DNA and evaluate the influence of Mg²⁺ on the binding between balofloxacin and DNA. MethodsFluorescent spectroscopy was used to study the interaction of balofloxacin with DNA and to calculate the thermodynamic constants. UV-Vis spectra, DNA viscosity titration, competition experiment and the effect of dsDNA and ssDNA on the fluorescense intensity were used to identify the binding mode. ResultsBalofloxacin interacted with CT-DNA with a quenching constant of (5.43±0.07)×10³ L·mol^-1. The interaction was exothermic with a Van’t Hoff enthalply of -8.03 kJ·mol^-1. Mg²⁺ cation could enhance the quenching constant between balofloxacin and DNA. ConclusionBalofloxacin interacted with CT-DNA in the mode of groove binding and Mg²⁺ could mediate the binding of balofloxacin to DNA .