In this study, we aimed to separate the enantiomeric forms of d,l-histidine, one of the essential amino acids, through molecular imprinted monolithic capillary electrochromatography columns prepared using hydrophobic N-methacryloyl-(l)-phenylalanine methyl ester as the functional monomer, and l-histidine as the template molecule. We investigated the effect of monomer ratio, temperature, template molecule mole ratio, crosslinker ratio, and porogen ratio to improve the permeability properties of the monolithic column. Characterization studies of the column were evaluated by attenuated total reflectance-Fourier transform infrared spectroscopy, scanning electron microscopy, and Brunauer-Emmett-Teller analysis. The chromatographic performance of the column was investigated using alkylbenzene-derived compounds. We evaluated some parameters to determine the optimum conditions for electrochromatographic studies such as electric field, organic solvent ratio, and pressure effect. The calculated imprinting factor (2.18) proved that the l-histidine imprinted amino acid-based monolithic column separated d,l-histidine molecules efficiently (percent relative standard deviation < 1.5) from each other using molecular imprinting technique with high-resolution values (resolution value > 1.5). As a result, selective d,l-histidine separation was achieved in less than 10 min at pH 7.0 without using a ligand or extra modification step for the first time.