The most crucial protein that affects the ability of soybean protein to emulsify is β-conglycinin. The subunit composition of β-conglycinin is one of the important factors affecting its emulsification performance. In order to investigate the effect of β-subunit of β-conglycinin on its emulsifying property, the separated and purified β-subunit was added to β-conglycinin in different proportions, and the properties of the β-conglycinin before and after emulsification were measured. The β-subunit appeared to improve the stability of the β-conglycinin in the aqueous dispersion as evidenced by the average particle size significantly decreasing, the surface hydrophobicity significantly increasing, and the Emulsification activity index and Emulsion stability index further improving. The secondary structure of β-conglycinin was altered by the addition of β-subunit, which caused more conversion of β-sheet and β-turn to α-helix, increasing the flexibility of protein molecules. Additionally, the particle size distribution, ζ-potential and creaming index of the stable emulsion formed by β-conglycinin were also measured. The results showed that the increase of β-subunit content improved the stability of the emulsion to some extent. The stability of emulsion mainly relies on their internal structure to maintain. It could be concluded from rheological analysis that the viscosity of emulsion increases due to the presence of β-subunits and the existence of weak gel structure inside them, which is corroborated by the results of confocal laser scanning electron microscopy. The elucidation of the influence of subunits on the emulsification characteristics of soyabean protein is beneficial to the development of soybean protein industry.