Chiral covalent organic frameworks (CCOFs) have potential application in enantioseparation due to their advantages, such as large surface area, abundant chiral recognition sites and good chemical stability in organic solvents. However, the application of CCOFs in high performance liquid chromatography (HPLC) for enantioseparation has been rarely reported because of the shortcomings of CCOFs, such as light weight, irregular shape, and wide particle size distribution. In order to overcome the above shortcomings, a one-pot synthetic method was adopted to prepare a core-shell composite (β-CD-COF@SiO2) via the growth of chiral β-CD COF on the surface of amino-functionalized SiO2 microspheres. The as-prepared β-CD-COF@SiO2 microspheres were used as a stationary phase for HPLC enantioseparation. The resolution ability of the β-CD-COF@SiO2-packed column toward various chiral compounds was investigated using n-hexane/isopropanol as the mobile phase. The results show that the chiral β-CD-COF@SiO2-packed column exhibited excellent chiral recognition ability for 24 pairs of chiral compounds with good reproducibility. These successful applications indicate that the preparation of the chiral COFs@SiO2 core-shell microspheres as a novel stationary phase for enantioseparation has good application prospects in HPLC.