Abstract: A method was developed to separate the enantiomers of ofloxacin, one kind of broad spectrum antibiotics, by capillary electrophoresis with derivatized cyclodextrins (CDs) as chiral selector. The separation was performed on a 57 cm×75 μm uncoated capillary. The optimal composition of mobile phase was 40 mmol·L^-1 DM-β-CD(dimethyl-β-cyclodextrin) in pH 2.5 phosphate buffer. The separation factor of enantiomers was 2.18. A diode array detector was employed for the detection of enantiomers. The effects of CD concentration in buffer, the electrolyte concentration, temperature and separation potential were also investigated.