A recyclable affinity ligand for immobilized metal affinity separation with temperature-induced effect was prepared using the appropriate synthesis route. The random copolymer of ethylene oxide and propylene oxide (EOPO) was chosen as the thermoseparating polymer. After suitable activation on EOPO the metal chelating function group (iminodiacetic acid, IDA) was coupled. The optimal conditions for preparing EOPO-IDA-metal were explored in the present work, and the Cull content in EOPO-IDA-Cu(II) could reach 0.70 mol mol(-1) EOPO. The results indicated that the couple of IDA and IDA-metal to EOPO has a little influence on the cloud point of EOPO. The affinity partitioning of nattokinase in the aqueous two-phase system demonstrated the effect of EOPO-IDA-metal on the selective partitioning of metal binding protein. Some factors, such as metal ions, EOPO-IDA-metal concentration, pH, amount of fermentation broth, and addition of metal chelating reagents, were investigated for optimizing the nattokinase separation. A two-step separation process combining immobilized metal ion affinity partition and temperature-induced phase separation was proposed to separate nattokinase directly from Bacillus subtilis fermentation broth, which utilizes the immobilized metal ion affinity partition for selective partitioning and the temperature-induced effect to achieve the recycling of phase-forming polymer and affinity ligand.