Abstract: Soybean protein is one of the major plant-derived protein resources due to its high yield and a wide range of processing applications. It is necessary to effectively improve processing properties of soybean protein, and thereby significantly enhance its nutritional value and economic potency. Most previous research mainly focused on the effects of external physical or chemical conditions and other complex system on the processing parameters of soybean protein. Few studies developed to explore the effect of various subunits compositions on the processing properties of soybean protein, particularly lacking on the deficiency of small subunit. Therefore, this study aims to investigate the effects of α and α′ subunits in β-conglycinin on emulsifying properties of soybean protein. In the experiment, some raw materials include Dongnong 47 and three different protein subunit-deficiency (α subunit-deficiency, α′ subunit-deficiency and α′, α subunit-deficiency) soybeans, to extract the soy protein isolates (SPI), by using the method of alkali solution acid precipitation for the preparation of emulsion. The subunit composition was then analyzed by sodium dodecyl sulfonate polyacrylamide gel electrophoresis (SDS-PAGE). Some parameters, including emulsifying activity index (EAI), ζ-potential, particle size, microstructure, turbiscan stability index (TSI), and interface adsorbed protein, were selected to evaluate the effect of α and α′ subunits in β-conglycinin (7S) on emulsifying properties of soy protein isolate. The results showed that the soy protein isolate emulsion of α′ subunit-deficiency has the highest emulsifying activity index (87.59 m2/g), the maximum ζ-potential absolute (47.7 mV), the minimum particle size (2.223 μm), the smallest molecules and most evenly distributed through the analysis of microstructure, the smallest turbiscan stability index (TSI), and the maximum content of interface adsorbed protein (31.40%), compared with other two SPI. The emulsion stability of soy protein isolate ranked in order: α′ subunit-deficiency, Dongnong 47, α subunit-deficiency, α and α′ subunit-deficiency. Safe and risk-free high-emulsifying products become an urgent demand because nutritious and healthy food has received increasing attention with the gradual improvement of people's living standard and the country's attention to the big health industry in recent years. Soy protein isolate, as a sort of amphiphilic structure, is commonly used in industrial production due to its high nutritional value and many functional characteristics. Therefore, emulsions can be served as cosmetic and pharmaceutical applications, as well in several food products, such as ice cream and mayonnaise with its different types. Since emulsions are an unstable system forming by water, oil, and emulsifiers, the instability of emulsion layer may reduce the shelf life of emulsion products. It was found that the addition of SPI with emulsifying properties can stabilize the emulsion in this study. The studies demonstrated that the α′ subunit-deficiency SPI obtained through breeding with Chinese genetic background can pose a great positive impact on the emulsifying properties of soybean protein, and thereby to effectively improve the quality characteristics of soybean protein isolate series products. This finding can also provide a theoretical and technical support for the development and application of high-emulsifying soybean protein series products. This study can offer a promising potential for intensive processing and cost-saving downstream processing, and thereby to meet the industry demands of “specific products, special varieties” in the national soybean industrial technology R&D system.