The interaction of ofloxacin (OFLX) and levofloxacin (L-OFLX) with calf thymus DNA(ctDNA)was studied by absorption spectra, fluorescence spectra, fluorescence polarization, and K3Fe(CN)6 quenching tests to show the interaction differences and binding modes. The experimental results of absorption spectra indicated that when ctDNA was added to the solution of OFLX or L-OFLX and its concentration was increased, the hypochromic effects in the absorption spectra of OFLX or L-OFLX were observed, and no changes in its maximum absorption wavelengths were found. Fluorescence spectroscopy was an appropriate method to study the interactions between small molecule ligands and biomacromolecule. From the measurements of emission peaks, transfer efficiency of energy, lifetime and so on, a vast amount of information about the interaction will be given. The experimental results indicated that the fluorescence quenching effects of OFLX and L-OFLX were not initiated by the dynamic collision according to the Stern-Volmer equation, and caused by the static quenching of compounds formation. From the Scatchard equation, its association constants were obtained for OFLX and L-OFLX to be 1.15×105 and 3.75×105 L?mol-1, respectively. On the basis of the absorption spectra and the association constants, it was showed that the interaction between L-OFLX and ctDNA was stronger than that of OFLX and ctDNA. There are three modes for the binding of small molecules to DNA double helix: electrostatic binding, groove binding and intercalative binding. The interaction modes between OFLX, L-OFLX and ctDNA were studied by fluorescence polarization, the K3Fe(CN)6 quenching tests and so on. The results showed that the interaction modes of OFLX and L-OFLX with ctDNA were both groove binding.