In this work, maltodextrin-modified magnetic microspheres Fe3O4@SiO2-Maltodextrin (Fe3O4@SiO2-MD) with uniform size and fine morphology were synthesized through a facile and low-cost method. As the maltodextrins on the surface of microspheres were combined with maltose binding proteins (MBP), the magnetic microspheres could be applied to enriching standard MBP fused proteins. Then, the application of Fe3O4@SiO2-MD in one-step purification and immobilization of MBP fused proteins was demonstrated. For the model protein we examined, Fe3O4@SiO2-MD showed excellent binding selectivity and capacity against other Escherichia coli proteins in the crude cell lysate. Additionally, the maltodextrin-modified magnetic microspheres can be recycled for several times without significant loss of binding capacity.