Based on the technology of protein separation with immobilized metal ion affinity chromatography (IMAC), a method for oriented immobilization of papain has been selected. Papain was successfully immobilized on magnetic agarose carriers. Cu2+ with iminodiacetate (IDA) was used as the chelating ligand to be correlated with the histidine on papain (His-81). The optimum immobilization conditions of enzyme were as flows: Cu2+ 0.15 x 10(-2) mol/g carriers, time was 4h, enzyme load was 30 mg/g carrier, pH was 7.0, respectively. The pH and temperature were 8.0 and 70 degrees C for immobilized enzyme. The recovery activity of immobilized enzyme was retained 68.4%. The carrier could be recovered from the spent immobilized enzyme, to be reused. After 5 times, the reimmobilization of papain on the regenerated matrix was 79.71% effective with the retention of maximum enzyme activity. The cost of carriers used for industrial applications is very important. The regenerability of carriers is therefore, relevant. The mild conditions used for immobilization, the high recovery of immobilized preparations, the stability and the regenerability of the matrix are the main features of the method reported here. All above indicate this method can be applicable and promising in enzyme immobilization field.