Phosphatidylcholine-soybean protein isolate (PC-SPI) nanoemulsions were prepared by ultrasonication. The effects of preparation conditions (SPI and PC addition, ultrasonic power and time) on the structural properties of the nanoemulsions and their storage stability were investigated. The results showed that the most optimal adsorption capacity and adsorption tightness at the oil-water interface under optimal conditions (1.5% SPI, 0.20% PC, 500 W ultrasonic power and 9 min ultrasonic time) were exhibited by the SPI-PC conjugate, which demonstrated that this nanoemulsions can be categorized as a high-quality emulsion suitable for research. To test its stability, and the high-quality nanoemulsion of β-carotene was stored. After degradation of the nanoemulsions during storage, β-carotene was released. The β-carotene retention rate of the high-quality emulsion was maintained above 86% at different temperatures in the absence of light for up to 30 days. This study provides new information for the development of transport and stability systems for nanoemulsions.