Abstract: Soybean peptide is a new type of multifunctional natural surfactant with biological activities such as nutrition, taste and antioxidant. In order to take better use of soybean peptide as emulsifier in food emulsions, soybean protein isolate hydrolysates (SPIH) were prepared by enzymolysis of soybean protein isolate, and SPIH-stabilized nanoemulsions were firstly prepared by ultra-high pressure homogenization (UHPH) in this study. The effects of emulsifier concentrations, homogenization pressure and cycle numbers on the average particle size, particle size distribution (PDI) and physical stability (Ke value) of nanoemulsions were explored. The three preparation conditions were optimized by orthogonal experiment compromising between the mean particle size and Ke value. Particle size analyzer and atomic force microscope (AFM) were applied to characterize the storage stability and microstructure of the nanoemulsion, respectively. Results showed that the optimal conditions were SPIH concentration 20 mg/mL, homogenization pressure 140 MPa and cycle numbers 5, while the nanoemulsion with particle size of 178.8 nm, Ke=7.37% was obtained under this condition, and stabilized for more than 56 d. AFM images showed that droplets of the nanoemulsion distributed uniformly and droplets aggregated when the peptide concentration increased. This study would provide a reference for the preparation and application of nanoemulsions with polypeptide emulsifiers.