A new type of polymethacrylate-based monolithic column with chiral stationary phase was prepared for the enantioseparation of aromatic amino acids, namely d,l-phenylalanine, d,l-tyrosine, and d,l-tryptophan by CEC. The monolithic column was prepared by in situ polymerization of butyl methacrylate (BMA), N-methacryloyl-l-histidine methyl ester (MAH), and ethylene dimethacrylate (EDMA) in the presence of porogens. The porogen mixture included DMF and phosphate buffer. MAH was used as a chiral selector. FTIR spectrum of the polymethacrylate-based monolith showed that MAH was incorporated into the polymeric structure via in situ polymerization. Some experimental parameters including pH, concentration of the mobile phase, and MAH concentration with regard to the chiral CEC separation were investigated. Single enantiomers and enantiomer mixtures of the amino acids were separately injected into the monolithic column. It was observed that l-enantiomers of aromatic amino acids migrated before d-enantiomers. The reversal enantiomer migration order for tryptophan was observed upon changing of pH. Using the chiral monolithic column (100 m id and 375 m od), the best chiral separation was performed in 35:65% ACN/phosphate buffer (pH 8.0, 10mM) with an applied voltage of 12 kV in CEC. SEM images showed that the chiral monolithic column has a continuous polymeric skeleton and large through-pore structure.