In order to develop new protein imprinting polymerization methods and to prepare protein imprinted nanomaterials with high recognition, a novel protein imprinted strategy is developed in this study by using polyethyleneimine (PEI) for aminolysis of tailor-made thiolactone-based functional monomers and crosslinkers on amine-modified magnetic nanospheres in an aqueous medium. The prepared protein imprinted nanospheres can seize BSA templates in the imprinting polymerization process through multiple hydrogen bonds, and hydrophobic and electrostatic interactions. In addition, the aminolysis reaction also generates abundant amide bonds in the imprinting polymer network, which not only enhances the hydrogen bonding interactions between the imprinted nanospheres and BSA but also improves the stability of the imprinting cavities by increasing the rigidity of the polymer chains. Based on the above advantages, the protein imprinted nanospheres show excellent rebinding specificity for BSA, for which the rebinding capacity is up to 505 ± 15 mg g-1 and the imprinting factor is 4.09. The protein imprinted nanospheres exhibit fast adsorption kinetics, outstanding reusability, and can selectively separate BSA from a protein mixture and actual biological samples. The generality of this imprinted method is also verified. Thus, this study will provide a new idea for the design of protein imprinted materials with high recognition.