In order to study the effect of different subunit compositions of β-conglycinin on the microstructure and functional properties of soy protein isolate film, Dong Nong 47 (Wild control), α-lack, α′-lack and (α+α′)-lack soybeans were selected as raw materials to extract soy protein isolate, and the subunit composition of four soy protein isolates was analyzed by sodium dodecyl sulfonate polyacrylamide gel electrophoresis. The soy protein isolate film composed of different subunits was prepared by the casting method, and the mechanism of its rheological properties, microstructure, mechanical strength, water resistance, barrier properties, thermal stability, and optical properties were studied. The results showed that the composition of different subunits had an effect on the microstructure and functional properties of the soybean protein isolate film. Compared with the deletion of α subunit and the deletion of α and α′ double subunits, the absence of α′ subunit had the most obvious impact on membrane structure and characteristics. The results of fourier transform infrared spectroscopy analysis showed that compared with Wild films, the deletion of α′ subunit enhanced the interaction of protein intermolecular hydrogen bonds. The contents of β sheets in the α′-lack film decreased to 27.84%, and the content of α helices and random coils increased to 16.83% and 13.84%, respectively. Scanning electron microscope results showed that the textures of α-lack and (α+α′)-lack films were more scattered and loose, while the α′-lack films had no obvious bubbles and had a flat and dense network structure. The results of rheological properties showed that the deletion of α subunit would reduce the film-forming strength, while the deletion of α′ subunit would significantly increase the G′ value of the membrane fluid and the potential film-forming ability of α′-lack. In comparison with the other three films, α′-lack film had the maximum tensile strength (2.31MPa), the minimum water content (9.54%) and the water solubility (30.81%), and the lowest water vapor transmission coefficient [21.89g·mm/(d·m²·kPa)], and showed excellent thermal stability and visible light-blocking properties. The results of the study found that the deletion of α′ subunit could significantly improve the microstructure and functional properties of soy protein isolate films, providing theoretical and technical support for the development of soybean protein products with high film-forming property.