Protein-imprinted polyacrylamide (PAM)-grafted calcium alginate (CaA) microspheres were synthesized via free-radical grafting polymerization with potassium persulfate and sodium bisulfite as initiators. FTIR, C-13-NMR and SEM were used for characterization. Swelling behaviors were studied and it was found that modified calcium alginate (CaA) microspheres possessed larger swelling degree than calcium alginate, because grafted hydrophilic PAM side chains contributed to more water absorption. Moreover, the durability of the swollen samples was improved for the modified microspheres. Compared with calcium alginate microspheres, modified calcium alginate samples exhibited higher absorption capacity (approximately, 13.1 and 10.3 mg/g) for MIPs in single and binary protein solution. The rebinding selectivity of CaA-g-PAM microspheres (approximately, 2.7) was higher than that of calcium alginate (CaA) microspheres (approximately, 1.5) in binary protein solution. It was suggested that the introduction of PAM side chains was beneficial for the specific recognition of protein.