Chiral polymer microspheres have a porous structure of a concentric multi-shell structure. Each layer of the multi-shell structure is optically and structurally anisotropic. The optical axes of adjacent layers have a sequential slight twist. All layers of the multi-shell structure generate a helix configuration and the chiral polymer microspheres are optically active. A method for preparing the chiral polymer microspheres, includes: forming a homogeneous liquid crystal mixture; dispersing the liquid crystal mixture into a continuous phase to form liquid crystal droplets through an emulsification process; polymerizing the reactive liquid crystal to form intermediate microspheres; and removing the one non-reactive liquid crystal and the chiral additive to form the chiral polymer microspheres. The chiral polymer microspheres have a porous structure and a swelling ability, and can be used as the stationary phase in chiral chromatograph, improving separation efficiency.