The binding mode and affinity of ofloxacin, which has two enantiomers (S and R-ofloxacin), to calf thymus DNA has been investigated by spectroscopic techniques in this work. When both enantiomers bind to DNA, the spectral properties in the drug absorption region was characterized by decrease of fluorescence intensity, hypochromism in the absorption and negative linear dichroism. The orientation angle of the transition moments of both enantiomers with respect to the DNA helix axis calculated from reduced linear dichroism were 60^o-70^o. These spectroscopic properties of enantiomers suggest non-classical intercalative binding from the minor groove for both ofloxacin enantiomers which is similar to be reported results for the norfloxacin-DNA complex (J. Am. Chem. Soc. (1998), 120, 6451-6457). Binding constant measured by Stern-Volmer method and Benesi-Hildebrand method of S ofloxacin is greater by five times compared to R-ofloxacin, indicating that the binding of S-ofloxacin to B-form DNA is far more favorable.