A ligand-exchange-based restricted access stationary phase was prepared via atom transfer radical polymerization. The stationary phase with the bilayer-polymer structure contains an inner layer for chiral separation and an outer layer for protein exclusion. L-Hydroxyproline was modified on the inner layer as a chiral selector, and the benzene rings on the diblock inner layer play an assistant role for chiral separation and improve the symmetry of the peak shape. Under HPLC method, the stationary phase exhibits well chiral separation ability and protein exclusion ability. The results demonstrate that the stationary phase can be potentially used for chiral separation with a direct injection of biological samples.