In this paper,taking the magnetic chitosan microspheres( M-CS) as the carrier,the effects of enzyme-to-carrier ratio,pH,glutaral concentration,reaction time on the activity and activity recovery of immobilized Alcalase were investigated. The characterizations and microstructure of immobilized enzyme were also studied. The results showed that the maximum activity of immobilized Alcalase reached( 86779 ± 119. 26) u /g and the activity recovery of immobilized Alcalase was( 77. 48 ± 0. 11) % under the conditions of enzyme-to-carrier ratio 112000 u /g,pH 8. 5,glutaral concentration 8%( V /V),and reaction time 11 h. The optimal reaction pH values for free and immobilized Alcalase were 11 and 10. 5 respectively. both The optimal reaction temperatures for free and immobilized Alcalase were 60 ℃. The pH and temperature stabilities of immobilized Alcalase were more excellent than those of free Alcalase. The activity of immobilized Alcalase reused 5 times remained 80. 89%( ± 0. 20%). According to Km value,immobilized Alcalase showed stronger affinity than free Alcalase. Transmission electron microscope showed that Fe3O4 and chitosan microspheres were all spherical nanopaticles with smooth surface,so more enzyme could be bound due to high specific area. FT-IR revealed that Fe3O4 was well wrapped up in M-CS,Vibrating sample magnetometer indicated that immobilized Alcalase had a good magnetic response.