A novel chiral stationary phase (CSP) of Zr-based metal-organic framework, l-Cys-PCN-224, was prepared by one-pot method and applied for the enantioseparation by capillary electrochromatography. The CSP was characterized by X-ray diffraction, thermogravimetric analysis, X-ray photoelectron spectroscopy, Fourier-transform infrared spectra, nitrogen adsorption/desorption, circular dichroism spectrum, zeta-potential, and so on. The results revealed that the CSP had good crystallinity, high specific surface area (2580 m2 /g), and good thermal stability. Meanwhile, the cross-section of l-Cys-PCN-224-bonded open-tubular (OT) column was observed by a scanning electron microscope, which proved the successful bonding of l-Cys-PCN-224 particles to the inner wall. Relative standard deviations of the column efficiencies were 3.87%-9.14%, and not obviously changed after 200 runs, which indicated that l-Cys-PCN-224-bonded OT column had the better stability and reproducibility. Excellent chiral separation performance was verified with nine kinds of natural amino acids including acidic, neutral, and basic as the analytes. All amino acids studied achieved good separation with the resolution of 1.38-13.9 and selector factor of 1.11-3.71. These results demonstrated that the CSP had an excellent potential in the chiral separation field.