Recombinant human Fab(rh Fab) with a hexahistidine tail attacked to the carboxyl end of Fd could be easily purified by immobilized metal ion affinity chromatography (IMAC). IMAC with different immobilized metal ions(Zn²⁺ and Cu²⁺) and different elution strategies (pH and imidazole gradient) were compared. The results showed that Cu²⁺ were more effective than Zn²⁺ in retaining the His tagged Fab protein. pH gradient showed better consistency in Fab recovery than imidazole gradient. Using Cu²⁺ IMAC more than 95% pure rh Fab could be obtained by one step purification.