The stability of soybean protein emulsion depends on the stability of protein membrane at oil-water interface. It is important to study the structure and functional property of interface protein of different protein emulsions. Totally 12 varieties of different interface protein were extracted. The interface flexible protein had higher content of hydrophobic amino acids, and the molecular weight of the protein contained mainly more than 100ku. The globulin subunits of 7S and 11S protein were analyzed by FTIR. The content of α-helix was lower and the content of random coil was higher, which was consistent with the above results of different flexible SPIs. From the composition of SDS-PAGE subunits, it could be seen that the proportion of 7S in the flexible protein was increased, which further proved that 7S protein had higher flexibility. The flexibility of the interface protein was higher. The result of surface hydrophobicity of interface protein was the same as that of SPI, and the interface protein had more hydrophobic residues to be stable at the water-oil interface. From the analysis of the tertiary structure of the interface protein by UV and fluorescence spectrum, it could be seen that the amino acid residues of the interface flexible protein were more exposed and the structure was more flexible, which was conducive to the molecular rearrangement at the interface. The functional properties were also changed, the solubility was decreased and the emulsification was increased, which was related to the structure features of the interface proteins.