Molecular recognition of proteins is critical for study and manipulation of protein-related biological processes. However, design and synthesis of abiotic receptors for precise recognition of proteins still remains a challenging task. Herein, we developed a universal sequential surface-imprinting strategy that integrated two different types of imprinting reactions to construct artificial protein receptors with high selectivity. Employing dopamine self-polymerization and boronate/diol complexation as the first-step and second-step imprinting reactions, respectively, we synthesized surface-imprinted magnetic nanocomposites against two different enzyme proteins: deoxyribonuclease I (DNase I) and apurinic/apyrimidinic endonuclease/redox effector factor 1 (APE1). The obtained nanocomposites both showed strong and specific binding toward their respective template proteins. Moreover, the bound enzymes could be totally recovered with high activity under mild buffer conditions. These antibody-like specific and reversible binding properties enabled effective purification and enrichment of the low-abundance target proteins from complex serum samples. Compared to existing one-pot or one-step imprinting methods, the proposed sequential surface-imprinting approach offers a more flexible combination of different functional monomers and greatly enhances the performance and biocompatibility of the imprinted materials. The generality and simplicity of the sequential imprinting strategy would make it an appealing and competitive method to prepare artificial protein receptors.