Abstract: Puroindoline,influenced and decided the wheat hardness and exhibiting activity against gram-positive bacteria,gram-negative bacteria and fungi,is a very important protein in wheat flour.In this studies,the puroindoline was extracted from wheat flour in the 100 mmol/L Tris-HCl pH7.8 buffer,containing 4% Triton X-114.Then the protein mixture including puroindoline was successively purified by Sephadex G-75,and each fraction was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE).Fractions containing polypeptides with apparent molecular weights of less than 31 kD were pooled and loading on Carboxymethyl-cellulose(CM-23) after concentrated by PEG20000,and each fraction was analyzed as preceding.In those ways,they were eluted by distilled water and applying a gradient from 0.05 mol/L to(0.7 mol/L) NaCl in 8 mmol/L MES pH5.5 buffer respectively.The fractions containing polypeptides apparent molecular weight 15 kD were pooled and concentrated by PEG20000,at last the puroindoline was obtained by freeze-dried.