［B10Glu,B25-Glu-B26］human proinsulin gene was obtained by asymmetry PCR and cloned into the expression vector pBV220 and expressed in Escherichia coli DH5α in the form of inclusion body.The target protein was refolded in vitro and purified. After lysis with trypsin and carboxypeptidase B, an insulin analogue was purified by DEAE ion exchange and RP-HPLC.The product was characterized by MALDI-TOF MS.Circular dichroism spectrum was studied. The results implied the changes of the secondary structure and the association.The association of the analogue was further studied by size exclusive chromatography with a strong monomeric property. The biology activities including RIA, RBA were assayed, exhibiting 63.5% and 114.4% those of native insulin respectively. The result of mouse convulsion test indicated the insulin analogue had in vivo activity nearly the same as native insulin.