Magnetic hydrogel microspheres mere prepared by co-polymerizing N-isopropylacrylamide (NIPAM), methacrylic acid (MAA) and N,N＇-methylene-bis-acrylamide (MBA) in the presence of ultrafine magnetite particles (average particle diameter of 10-20 nm) prepared by co-precipitation. The synthesized magnetic hydrogel microspheres with varying MAA content were monodispersed, and their average diameters ranged from 150-250 nm. All the magnetic hydrogel microspheres showed a reversible transition between flocculation and dispersion as a function of temperature (4-40 degrees C), and the thermo-flocculated microspheres were separated quickly in the magnetic held. Enzymes were immobilized onto these thermo-sensitive magnetic hydrogel microspheres by two different methods. Trypsin was covalently immobilized onto the magnetic hydrogel microspheres in high yield by the carbodiimide method. In the case of microspheres with higher MAA content, immobilized trypsin demonstrated higher activity. On the other hand, a fusion enzyme consisting of affinity tag AG (immunoglobulin G binding domains) and beta-galactosidase (beta gal) was immobilized onto the magnetic hydrogel microspheres with covalently immobilized human gamma-globulin by affinity interaction. The immobilized AG beta gal retained high activity even in the case of microspheres with a low MAA content. Therefore, the thermosensitive magnetic hydrogel microspheres are useful carriers for enzyme immobilization.