A restricted access media-molecularly imprinted polymer (RAM-MIP) for propranolol (PRP) has been prepared for direct injection analysis of β-blockers in biological fluids. First, the MIP for PRP was prepared using methacrylic acid and ethylene glycol dimethacrylate as the functional monomer and cross-linker, respectively, by a multi-step swelling and polymerization method. Next, a 1:1 mixture of glycerol monomethacrylate and glycerol dimethacrylate was used for hydrophilic surface modification, and added directly to the MIP for PRP after 4 h from the start of polymerization. Then further polymerization was carried out for 20 h. The obtained RAM-MIP for PRP showed excellent molecular recognition ability for PRP, good ones for alprenolol (ALP) and pindolol, and fair ones for other β-blockers. The RAM-MIP was applied for direct injection analysis of ALP enantiomers in a rat plasma sample by a column-switching HPLC system using a β-cyclodextrin phenylcarbamate-bonded silica column as the analytical column. The calibration graph, constructed from peak area versus each ALP enantiomer concentration, was linear with a correlation coefficient of >0.999 over the concentration ranges of 12.5–250 ng ml⁻¹. The limit of quantitation was 12.5 ng ml⁻¹ with a 50 μl injection. This method could be applicable for the assay of ALP enantiomers at the therapeutic plasma levels, and have wide applicability for the assay of β-blockers in biological fluids.