A novel mode of chiral separation in electrochromatography (CEC) with dynamically modified stationary phase (DMS-CEC) was presented. The capillary column was packed with strong anionic exchange stationary phase, the sulfated β-cyclodextrin (S-CD), which was added in the mobile phase, dynamically adsorbed to the packing surface and a new layer of chiral stationary phase was formed. The separation of enantiomer was based on their different interaction with the new stationary phase. The enantionmers of tryptophan, atropine and verapamil were successfully separated in this system with resolution of 2.06, 10.1 and 1.96, and the column effeciency for the enantiomers were varied from 85000 plates/m to 412000 plates/m. The relative standard deviation (RSD) of void time and the tryptophan enantiomers′ migration time for 17 consecutive runs were 0.5%, 0.6% and 0.7%, respectively. Enantiomer separation by capillary electrochromatography with adsorbed bovine serum albumin (BSA) and sulfated cyclodextrin (S-CD) as chiral stationary phases were also studied. The resolution for tryptophan enantiomers in the two systems were 3.86 and 2.97, respectively. It was found that the superiority of DMS-CEC over the adsorbed S-CD column CEC was that better repeatability could be obtained in DMS-CEC.