Abstract: The commercial soy protein isolate (SPI) was used as raw material to prepare soy protein nanoparticles (SPNPs) through either single enzymatic treatment or homogenization assisted with enzymatic treatment. The structural characteristics of particle size, polydispersity index, morphology, Fourier transform infrared and endogenous fluorescence spectra of SPNPs, as well as the physicochemical properties of the pattern of intra-particle interactive forces, surface hydrophobicity, Zeta-potential, amphipathy, emulsifying, and foaming properties of SPNPs were comparably analyzed. It was found that the SPI with large particle size (230.00 nm), the SPNPs prepared by either enzymatic or homogenization assisted with enzymatic treatment at low degree (3%) of hydrolysis (DH) were spherical and showed smaller size distributions with z-average size from 64.20 to 144.80 nm. The analysis of secondary structure implied that SPNPs prepared by homogenization assisted with enzymatic treatment showed an increased ratio of α-helix/β-sheet to a narrow range of around 45%. Compared with SPNPs prepared by single enzymatic treatment, the SPNPs showed a stronger negative charge (−33 mV) under neutral conditions and higher H₀, suggesting better emulsifying and foaming properties of the present SPNPs. Meanwhile, the results of the interactive force indicated that the hydrophobic interactions mainly dominated the structure formation of SPNPs, along with hydrogen bonds and disulfide bonds mainly stabilizing the external and internal structure of nanoparticles, respectively. The above results indicated that homogenization assisted with enzymatic treatment provided new solutions for the green preparation of multifunctional protein nanoparticles.