Enantioseparation of tryptophan enantiomers (Trys), tyrosine enantiomers (Tyrs) and phenylamine enantiomers (Phes) by chiral ligand exchange capillary electrophoresis (CLE- CE) using 1-ethyl-3-methyl imidazole L-lactate ([EMIM] [L-Lac ]) as the chiral ligand was studied. The effects of background electrolytes and central ions, the molar ratio and concentration of the chiral ligand and central ion, running buffer solution pH on the enantioseparation of Trys, Phes and Tyrs were investigated. Good enantioseparation of three pairs of enantiomers were obtained with 40.0 mmol/L [EMIM] [L-Lac] and 20.0 mmol/L cupric chloride (pH 4.5). To validate the good behavior of [EMIM] [L-Lac], L-lactate (L-Lac) was further used as the chiral ligand for the enantioseparation of Trys. The contrast experiment showed that Trys could only be partially enantioseparated by L-Lac. Moreover, the enantioseparation was improved by adding 1-ethyl-3-methyl imidazole acetate (EMIM-Ace) which markedly prolonged the migra- tion time of Trys to more than 30 min. In contrast, the migration time of Trys was obtained in 15 min by [EMIM] [L-Lac]. Finally, the enantioseparation mechanism was further discussed. The experimental results showed that in the EMIM-Ace assisting L-Lac system, EMIM-Ace was only used to suppress the electroosmotic flow and was not involved in the chiral ligand exchange reaction, while in the [EMIM] [L-Lac] system, the chiral ligand taking part in the re- action was mainly unionized [EMIM] [L-Lac] rather than L-Lac formed by ionized L-Lac ions.