An open tubular molecule imprinted polymer (OT-MIP) capillary column has been prepared for chiral separation of ofloxacin enantiomers in CEC. The S-ofloxacin imprinted OT column was fabricated by thermally initiated non-covalent polymerization procedure inside a pretreated and silanized fused silica capillary. The template molecule was incorporated with methacrylic acid (MAA), ethylene glycol dimethacrylate (EDMA) and 4-styrenesulfonic acid (4-SSA) and dissolved in a porogen mixture of ACN/2-propanol (9:1). The separation efficiency of the 4-SSA MIP column was found quite better than that of the MIP column without 4-SSA. It has been demonstrated that our OT-MIP column can separate ofloxacin enantiomers with excellent chiral separation efficiency after tuning the various chromatographic conditions. The optimized chromatographic eluent was 85:15, v/v%, ACN/60 mM sodium acetate at pH 7. The separation efficiency and selectivity of chiral separation of this study were far better than those obtained by previous methods for chiral separation of R- and S-ofloxacin.