•Hydrophilic microparticles were prepared via one-pot synthesis using RAFT precipitation polymerization.•The hydrophilic chains on the surface of microparticles can well improve their surface hydrophilicity and lead to their biological compatibility.•The microparticles can be used as chiral restricted access material (RAM).•As chiral RAM, the hydrophilic microparticles were used for determination of enantiomers in biological samples with direct injection via HPLC analysis.Novel hydrophilic microparticles containing β-cyclodextrin (β-CD) were prepared via one-pot synthesis using reversible addition-fragmentation chain-transfer (RAFT) precipitation polymerization, a “controlled/living” radical polymerization technique. The polymerization was initiated by hydrophilic macromolecular chain-transfer agent [poly(2-hydroxyethyl methacrylate), PHEMA]. The hydrophilic PHEMA on the surface of microparticles can well improve their surface hydrophilicity and lead to their biological compatibility. As chiral restricted access material (RAM), the hydrophilic microparticles can be used for determination of enantiomers in biological samples with direct injection via HPLC analysis.