Abstract: Human glutathione-S-transferase isozyme pi 1 (hGSTP1) is a target to tackle resistance of tumor. To explore an immobilization method suitable for affinity-drive-exponential-enrichment through magnetic separation for the screening of high-affinity ligands of hGSTP1 from mixtures, immobilizations of hGSTP1 on zwitterion-coated magnetic beads functionalized with Ni2+-NTA and carboxyl were compared. The recombinant expressions of 6×his-hGSTP1 were purified by Ni2+-NTA agarose medium and GSH-Sepharose 4B column, respectively.The immobilization capacity, retention activity, stability and the response of immobilized hGSTP1 to inhibitors were characterized. Carboxyl-functionalized magnetic beads showed undetectable immobilization of hGSTP1 purified with GSH-Sepharose 4B column, but manifest immobilization of hGSTP1 purified with Ni2+-NTA agarose, yielding only ~30% retention activity of the free hGSTP1. While hGSTP1 purified with both media were immobilized on Ni2+-NTA magnetic bead with the retention activity of ~80% and the saturation immobilization capacity 1.7 times of that on carboxyl-functionalized magnetic beads. The immobilized hGSTP1 on Ni2+-NTA magnetic beads under stirring in PBS (pH 6.5) at 0 ℃ for 8 h showed insignificantly change in activity, while the free enzyme activity decreased about 20%; the further treatment with methanol at 80 ℃ for 20 minutes resulted negligible protein released, supporting its stability to the screening and the subsequent treatment. There was no significant difference in IC50 to Ethacrynic Acid of hGSTP1 immobilized on Ni2+-NTA comparing with that of free enzyme (P>0.05). Therefore, the immobilization of hGSTP1 on Ni2+-NTA magnetic beads is suitable for affinity-drive-exponential-enrichment of hGSTP1 inhibitors in the mixture for screening.