The invention relates to the technical field of preparation and application of chiral high-performance liquid chromatography column materials, and discloses preparation and application of beta-CD-D2 high-performance liquid chromatography chiral column material. A high-performance liquid chromatography chiral column is prepared from beta-CD-D2 and is used for splitting chiral medicine enantiomers. The preparation and the application of the beta-CD-D2 high-performance liquid chromatography chiral column material comprise the following steps: 1, constructing a chiral environment by using beta-CD-D2, bonding the chiral environment with high-performance liquid chromatography silica beads into a fixed phase filler, then preparing the fixed phase filler into a chiral column, and applying the chiral column into high-performance liquid chromatography; 2, finding out an optional formula and an optional condition by using a proper characterization means, and optimizing the method and the condition; 3, establishing a preparation method of a salmeterol single enantiomer by using the prepared beta-CD-D2 chiral column, and splitting a chiral compound by using a cyclodextrins derivative for the first time; and 4, splitting drugs by using the prepared beta-CD-D2 chiral column, and establishing a method for splitting and qualitatively and quantitatively analyzing seven chiral drugs including salmeterol, terbutaline, procaterol, cetirizine, lamivudine, cefuroxime and ceftriaxone.