Abstract: In this paper, the effect of ultrasound technology on the structure of high concentrations soybean protein isolate (SPI) and its enzymatic hydrolysate antioxidant activity were explored. SDS-PAGE, Fourier transform infrared spectrum, fluorescence spectrum and fluorescence probe were used to analyze the effect of ultrasound on the molecular structure of soybean protein isolate at high concentrations, as well as on the content and molecular weight of polypeptides, free amino acid composition and antioxidant activity of enzymolysis products. The results showed that under certain ultrasound treatment conditions, the primary structure of SPI remains unchanged, but the high concentration (16%) of SPI changed other structural properties after ultrasound treatment. The β-sheet content increased and the α-helix content decreased in the secondary structure. In addition, the endogenous fluorescence emission wavelength was significantly redshifted, and the surface hydrophobicity was significantly (P<0.05) enhanced, which in turn had an impact on the effect of enzymatic hydrolysis SPI. As the SPI concentration increased, compared with no ultrasound treatment, ultrasound treatment increased the peptide content in the enzymatic hydrolysis products. At a concentration of 16%, the peptide content reached as high as 84.27%, and at a concentration of 8%~16%, the proportion of peptides with a molecular weight of 200~2000 Da increased. Furthermore, the levels of free hydrophobic amino acids in SPI enzymatic hydrolysates were increased by ultrasound treatment significantly (P<0.05), and at a concentration of 16%, the DPPH free radical scavenging rates and hydroxyl free radical scavenging rates of enzymatic hydrolysates were significantly improved, and the reduction ability was significantly enhanced, demonstrated higher antioxidant activity. It could be seen that the modification of ultrasound treatment on high concentrations SPI had contributed to prepare enzymatic hydrolysates with high antioxidant activity, which would have good application prospects in the preparation of peptides.