Anovel application of chiral ligand-exchange capillary electrophoresis (LE-CE) was developed with polymeric nanoparticles as a chiral ligand and Cu 2+ as a central ion. Nanoparticles (NPs) were prepared by polymerization of N-methacryloyl-L-histidine methyl ester (MAH) and ethylene dimethacrylate (EDMA). NPs were characterized by elemental analysis, fourier transform infrared spectroscopy (FTIR), atomic force microscopy (AFM). Average particle size and size distribution of NPs were also performed. Elemental analysis of MAH for nitrogen stoichiometry was found as 0.2 mmol/g polymer. CE systems that contain NPs in running buffer can be thought as pseudocapillary electrochromatography. Using this approach, enantiomer separation of ofloxacin was carried out by using LE-CE. The results demonstrated that NPs with chiral functionalized group interacted differently with structural enantiomers of ofloxacin. Factors affecting chiral resolution were studied. The optimum running conditions for the enantioseparation of ofloxacin were found to be a background electrolyte (BGE) (pH 4.7) containing 70% ACN, 10 mM CuSO4, 40 mM (NH4)2SO4 and 30 mg/mL NPs. Under these conditions, the enantioseparation of ofloxacin was successfully achieved. With this system, R-ofloxacin and S-ofloxacin (levofloxacin) were used to analysis capsules in the ofloxacin tablets.