The development of highly enantioselective assays and sensors has received much attention for the determination of enantiomeric impurities at a low level. For chiral compounds, the efficient monitoring of the in selection procedure has allowed the isolation of nucleic acid aptamers which are able to strongly discriminate the target enantiomers. In this paper, we demonstrated for the first time that an aptamer can be successfully used to design a highly enantioselective tool for the trace enantiomer detection. The aptamer-based stereoselective assay was developed using an affinity capillary electrophoresis-based competitive, homogeneous format and an on-capillary mixing approach. Detection of as low as 0.01% of the minor enantiomer in a nonracemic mixture can be achieved, in a short analysis time (<5 min).