A multi-template imprinted microchannel was prepared by a one-pot in situ imprinting process. The imprinted microchannel led to a novel chip-based strategy for simultaneous multiple enantioseparation. The one-pot imprinting process formed a multi-template imprinted porous thin layer (about 2 μm) on the inner wall of the capillary, which was characterized by scanning electron microscopy, infrared spectroscopy, and solid-state UV-vis spectroscopy. By fixing the imprinted capillary to a support substrate composed of poly(dimethylsiloxane) on a glass slide, a multi-analyte microchip was thus conveniently constructed. Using L-tyrosine (L-Tyr) and L-tryptophan (L-Trp) as the template molecules, two pairs of enantiomers were simultaneously baseline separated in a 6 cm separation channel within 120 s under the optimized preparation and electrochromatographic conditions. The separation showed excellent efficiency. The linear ranges for amperometric detection of four analytes using a carbon fiber microdisk electrode at +1.2 V (vs. Ag/AgCl) were from 20 to 500 μM for racemic Tyr and Trp. This multi-template imprinting strategy could be expanded for simultaneous separation and detection of additional pairs of enantiomers within a short analytical time. It could open up a promising avenue for high-throughput screening of chiral compounds.