Monolithic molecularly imprinted columns were designed and prepared by anin-situ thermal-initiated copolymerization technique for rapid separation of tryptophan andN-CBZ-phenylalanine enantiomers. The influence of polymerization conditions and separation conditions on the specific molecular recognition ability for enantiomers and diastereomers was investigated. The specious molecular recognition was found to be dependent on the stereo structures and the arrangement of functional groups of the imprinted molecule and the cavities in the molecularly imprinted polymer (MIP). Moreover, hydrogen bonding interactions and hydrophobic interactions played an important role in the retention and separation. Compared to conventional MIP preparation procedures, the present method is very simple, and its macroporous structure has excellent separation properties.