In previous studies, we synthesized the magnetic core-shell structured Fe3O4/PMG/IDA-Ni2+ nanoparticles. The Ni2+ on the surface of nanoparticles provides abundant docking sites for histidine, and the composite nano particles showed potential applications in the separation and purification of histidine-tagged (His-tagged) proteins. Meanwhile, the presence of the superparamagnetic core (Fe3O4) in the nanoparticles allows them to be quickly separated and purified by an external magnetic field. Herein, the ability of magnetic nanoparticles to purify His-tagged human superoxide dismutase 1 (hSOD1) was verified. SDS-PAGE and activity data showed His-tagged hSOD1 specifically bound to Fe3O4/PMG/IDA-Ni2+, and there was no significant competition for binding between final and three intermediate products. The binding capacity of nanoparticles can reach to 62.0 mg/g (dry weight of hSOD1/nanoparticles). The nanoparticle-bound hSOD1 exhibited better thermal and storage stability compared to free hSOD1. Furthermore, the purification efficiency of the magnetic nanoparticles in the separation and purification of His-tagged proteins was comparable to the other two commercial materials (High Affinity Ni-NTA Resin, HisPur Ni-NTA Magnetic Beads). Finally, the magnetic nanoparticles can be reused in the binding of His-tagged protein for multiple times. In conclusion, the nanoparticles are ready to be applied in the separation and purification of His-tagged protein.