A novel magnetic chitosan microsphere (MCM) was prepared from chitosan and Fe3O4 through inverse suspension crosslinking, epichlorohydrin activation, and grafted with ligands. The magnetometer measurements data indicated that the magnetic microspheres had superparamagnetic property as well as fast magnetic response without the magnetic hysteresis effect and thus can be well used in magnetic separation. The adsorption results showed that the magnetic chitosan microsphere grafted with ligand had good adsorption capacity on endotoxin up to 554.1 EU/g under the condition of 25.0 mg/mL adsorbent, 17.2 EU/mL endotoxin. It is expected that this new microsphere will have a feasible and useful application in adsorbing endotoxin from biological products.