Abstract: The recombinant cDNA of Tilapia Hsp70 was amplified from blood mRNA and inserted into vector pMD18-T.Sequencing revealed that the cDNA was subcloned into expression vector pPIC9K.The recombinant vector was transformed into the yeast Pichia pastoris KM71 via electroporation after sequencing.The transforming positive clones were screened by PCR and rtHsp70 in culture supernatant induced by methanol was identified by Western blot.Protein purification by the affinity chromatograph and gel filtration showed the sequence of rtHsp70 DNA was correct.It was a protein with relative molecular mass of 70 000 in the culture supernatant by SDS-PAGE and Western blot analysis.Two 2 mg purified rtHsp70 can be obtained in per liter expression supernatant,and Pichia pastoris engineer bacteria of rtHsp70 expression and purified protein were gained.The study is paved the way for further study of using rtHsp70 for immunologic adjuvant in peptide-vaccine application.