Trypsin (TRY) molecularly surface-imprinted material was prepared by synchronous grafting polymerization and surface imprinting with crosslinked polyvinyl alcohol (CPVA) microspheres as matrix. The reaction between methyl acryloyl chloride and the hydroxyl groups on the surface of CPVA microspheres occurred quickly, resulting in the modified MAO-CPVA microspheres with a great deal of polymerizable double-bond methylacryloyl (MAO) group. Anionic monomer sodium p-styrene sulfonate (SSS) and template TRY in a certain molar ratio were added in an aqueous solution, interacting each other. And the crosslinker N, N'-methylene bisacrylamide (MBA) was further added, and the modified MAO-CPVA microspheres were dispersed in the aqueous medium, the free radicals were produced by ammonium persulfate and sodium bisulfite redox initiating system, the graft-crosslinking polymerization of SSS surrounded around the template TRY and crosslinker MBA was conducted so that TRY surface-imprinted material MIP-PSSS/CPVA microspheres were obtained, and characterized. Moreover, their macromolecular recognition characteristics were studied. The results show that the MIP-PSSS/CPVA microspheres possess excellent binding affinity and specific recognition selectivity for the template TRY. The adsorption capacity of TRY on the microspheres reaches up to 85.9 mg/g, and the selectivity coefficient of imprinted TRY in contrast to protein lysozyme is up to 17.52.