The distribution behavior of ofloxacin enantiomers was examined in a biphasic system containing beta-cyclodextrin in aqueous phase and a complex formation of O'O-dibenzoyl-(2R, 3R)-tartaric acid (L-DBTA) and di(2-ethylhexyl)phosphoric acid in organic phase as the mixed extractants. The influences of pH, initial concentration of ofloxacin, phase ratio, and concentration of extractants were studied. The experimental results showed that both the distribution ratio and enantioselectivities of ofloxacin enantiomers were greatly improved. A higher enantioseparation efficiency with a maximum separation factor of 2.48 and enantiomeric excess of 40.4% can be obtained under the optimal experimental conditions. It could be very helpful to achieve high effective enantioseparation using the mixed extractants, which would be the guidelines for industrial amplification of chiral extraction separation.