A novel, simple, reproducible method was reported in this study for the enantioseparation and determination of DL-Tryptophan (DL-Trp) by using its corresponding DNA aptamer and partial filling technique in combination with micellar electrokinetic chromatography (MEKC).Aptamer was used as a chiral selector.Sodium cholate (SC) was used to form the chiral micelles and to enhance the enantioseparation of the enantiomers.Effects of aptamer concentration, filling time, buffer composition and separation voltage on the effectiveness of the enantiomers separation were evaluated.Under the optimal condition, D-and L-Trp were completely enantioseparated in less than 9 min.This Implementation of aptamer-based enantioseparations assays constitutes low-cost, low-complexity and better resolution on enantioseparation and it can be extend the aptamer-based enantioseparations in CE when corresponding aptamer used.