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.　©　2012　WILEY-VCH　Verlag　GmbH　&　Co.　KGaA,　Weinheim.