Abstract: In this study,soybean protein isolate( SPI) was extracted by alkali extraction and acid precipitation from natural selenium- enriched soybean,and SPI was sequentially precipitated by 40%,60%,and 80% of ammonium sulfate to obtain three protein fractions,which were 40%- SPI,60%- SPI,and 80%- SPI. In order to explore ways of selenium- enriched SPI fractionation by ammonium sulfate,the differences among the protein fractions were compared.The results showed that 60%- SPI had highest protein purity and selenium content.CD spectroscopy and SDS- PAGE results showed that the secondary structure and subunit composition were significantly different among the fractions.Through X- ray diffraction and atomic force microscopy,it found that crystal morphology and surface protein composition of the three fractions also varied widely. Therefore,ammonium sulfate salting precipitation was an effective primary screening method for separating selenium- enriched SPI with difference components,and 60% ammonium sulfate concentration was preferred.