Purification and refolding of bacterially expressed anti HBsAg ScFv by means of in situ refolding on column were investigated. Three refolding approaches were adopted: dialysis, in situ refolding via metal chelated chromatography and via gel filtration chromatography. The relative refolding index and relative recovery of the ScFv inclusion body were compared using the three procedures. The results showed that the refolding on gel filtration column(sephacryl S-200) was far more efficient than the other two approaches, with relative refolding index of 98% and relative recovery of 81%, illustrating the potential of this technique in production of recombinant ScFv.