In this study, a simple and reproducible method for enantioseparation and determination of dl-tryptophan (dl-Trp) was developed by using a partial filling technique in combination with MEKC. The corresponding l-Trp specific DNA aptamer was used as a chiral selector. Sodium cholate 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 enantioseparation were evaluated. The Mg2+ and Na+ concentration in separation buffer was found to effectively affect the separation efficiency and reproducibility. Under the optimal conditions, d- and l-Trp were completely enantioseparated in less than 9 min. This aptamer-based partial-filling approach has the potential to be extended to the separation of other enantiomers after the replacement of corresponding specific aptamers.