A new method for synthesis of an Immobilized-Metal Affinity chromatography (IMAC) adsorbent with superparamagnetism (Fe3O4/SiO2-GPTMS-Asp-Co) was reported in this paper. Fe3O4 nanoparticles were first modified by SiO2 to form the core-shell Fe3O4/SiO2 with superparamagnetism, the core-shell microspheres were then successively treated by 3-glycidoxypropyltrimethoxysilane (GPTMS), L-aspartic acid (L-Asp) and 2-bromoacetic acid to form Fe3O4/SiO2-GPTMS-Asp nanoparticles with tetradentate ligands. Finally, the IMAC adsorbent with superparamagnetism, Fe3O4/SiO2-GPTMS-Asp-Co, was finally obtained by the coordination of Co2+ with the resulting nanoparticles. The intermediates and product obtained from the process mentioned above were characterized by TEM, SEM, XRD, XPS, FT-IR, TGA, MS, EDS, element analysis and magnetic hysteresis loop. Fe3O4/SiO2-GPTMS-Asp-Co nanoparticles used as IMAC adsorbent to separate the recombinant 6-histidine-tagged gp41 protein were investigated. The result indicates that the IMAC adsorbent we prepared has outstanding advantages in the separation of the 6-histidine-tagged proteins from the crude bacterial lysate, such as simple operation, high selectivity and capacity. (C) 2010 Elsevier B.V. All rights reserved.