A microfluidic device integrated with molecularly imprinted magnetic nanoparticles as stationary phase was designed for rapid enantioseparation by capillary electrochromatography. The nanoparticles were synthesized by the co-polymerization of methacrylic acid and ethylene glycol dimethacrylate on 3-(methacryloyloxy)propyltrimethoxysilane-functionalized magnetic nanoparticles (25-nm diameter) in the presence of template molecule, and characterized with infrared spectroscopy, thermal gravimetric analysis, and transmission electron microscope. The imprinted nanoparticles (200-nm diameter) could be localized as stationary phase in the microchannel of microfluidic device with the tunable packing length by the help of an external magnetic field. Using S-ofloxacin as the template molecule, the preparation of imprinted nanoparticles, the composition and pH of mobile phase, and the separation voltage were optimized to obtain baseline separation of ofloxacin enantiomers within 195s. The analytical performance could be conveniently improved by varying the packing length of nanoparticles zone, showing an advantage over the conventional packed capillary electrochromatography. The linear ranges for amperometric detection of the enantiomers using carbon fiber microdisk electrode at +1.0 V (vs. Ag/AgCl) were from 1.0 to 500 microM and 5.0 to 500 microM with the detection limits of 0.4 and 2.0 microM, respectively. The magnetically tunable microfluidic device could be expanded to localize more than one kind of template-imprinted magnetic nanoparticles for realizing simultaneous analysis of different kinds of chiral compounds.