Summary: Developing high-performance materials for efficient isolation of proteins is of great importance. Herein, the authors synthesized a series of magnetic Fe,O,@PS-ANTA-M, (M = Ni, Co, Cu and Zn) nanospheres, consisting of fine Fe,O, nanoparticles encapsulated by functionalized polystyrene shell, where metal ions were complexed by Nα, Nα-Bis(carboxymethyl)-L-lysine hydrate for selectively immobilizing histidine (His)-tagged proteins. The structures of the Fe,O,@PS-ANTA-M, were studied by field scanning electron microscopy, transmission microscopy, Fourier transform infrared spectroscopy, thermogravimetric analysis, X-ray photoelectron spectroscopy and vibrating sample magnetometer. The adsorption performance of the Fe,O,@PS-ANTA-M, was studied using bovine hemoglobin as a model protein. The thermodynamics of the binding process was studied by isothermal titration calorimetry tests. The resulting Fe,O,@PS-ANTA-Cu,, Fe,O,@PS-ANTA-Ni,, Fe,O,@PS-ANTA-Co, and Fe,O,@PS-ANTA-Zn, possessed adsorption capacities of 21200, 8080, 6640 and 5360 mg g,, respectively, rendering them as the best adsorbents for protein purification. The adsorption isotherm was better fitted by Langmuir equation. To verify the selectivity, the Fe,O,@PS-ANTA-M, was practically employed to isolate His-tagged proteins from cell lysate. The results indicated that the samples showed outstanding adsorption capacity, selectivity and stability, and facile regeneration and separation, outperforming a commercial NTA-Ni column. These findings suggested that the Fe,O,@PS-ANTA-M, showed promising applications for specifically isolating proteins from complex biological systems.