Due to unique structural characteristics and chiral environment, β‐cyclodextrin (β‐CD) has a strong chiral separation ability. 4‐Chlorophenyl isocyanate (4CPI) also plays an important role in separating chiral drugs. In this paper, a chiral stationary phase: 4‐CPI‐β‐CD stationary phase (4CPI‐β‐CD@PMMA) was designed and prepared. Polymethyl methacrylate (PMMA) microspheres with uniform particle size and good monodispersity were prepared by modified seed polymerization and seed expansion methods. Using diazo resin as a coupling agent, PMMA microspheres were connected with 4CPI‐β‐CD to prepare a stationary phase with chiral recognition ability. PMMA microspheres and 4CPI‐β‐CD were also characterized. Compared with the traditional silica gel column, PMMA microspheres have higher preparation efficiency, better monodispersity, and better mechanical properties as chromatographic fillers. Finally, the chiral separation of ketoprofen was carried out by reversed‐phase high‐performance liquid chromatography, and the separation conditions were optimized. This paper is a good idea for the preparation of the chiral stationary phase.