This study investigates the effects of ultrasound-assisted L-histidine (L-His) treatment on the physicochemical properties and conformation of soybean protein isolate (SPI). The combination of ultrasound and L-His treatment significantly reduced the particle size, increased solubility, and decreased surface hydrophobicity of SPI. Spectral analysis revealed that L-His altered the secondary structure and three-dimensional conformation of SPI, reducing α-helix content and increasing zeta potential and active sulfhydryl content. At a concentration of 0.3% L-His, SPI exhibited optimal emulsification and antioxidant properties. The study concludes that ultrasound-assisted L-His treatment effectively modifies SPI, enhancing its functional properties, which has potential applications in soy protein processing and utilization.This study investigates the effects of ultrasound-assisted L-histidine (L-His) treatment on the physicochemical properties and conformation of soybean protein isolate (SPI). The combination of ultrasound and L-His treatment significantly reduced the particle size, increased solubility, and decreased surface hydrophobicity of SPI. Spectral analysis revealed that L-His altered the secondary structure and three-dimensional conformation of SPI, reducing α-helix content and increasing zeta potential and active sulfhydryl content. At a concentration of 0.3% L-His, SPI exhibited optimal emulsification and antioxidant properties. The study concludes that ultrasound-assisted L-His treatment effectively modifies SPI, enhancing its functional properties, which has potential applications in soy protein processing and utilization.