This study investigates the effects of ultrasound-assisted L-histidine (L-His) modification on the physicochemical properties and conformation of soybean protein isolate (SPI). The results show that ultrasound treatment reduces the particle size, increases solubility, and improves the homogeneity of SPI. The addition of L-His further reduces particle size, increases zeta potential, and enhances solubility and functional properties. Spectral analysis indicates that L-His forms a complex with SPI, altering the microenvironment of amino acid residues and improving emulsification and antioxidant properties. At a concentration of 0.3% w/w, the nanocomplex exhibits a smaller particle size (140.03 nm), higher zeta potential (-23.63 mV), and higher emulsification stability (22.48 min). The study also reveals that ultrasound and L-His treatment alter the secondary structure of SPI, reducing alpha-helix content and changing the microenvironment of hydrophobic amino acids. The optimal emulsification and antioxidant properties of SPI were achieved at a L-His concentration of 0.3%. The combination of ultrasound and L-His treatment effectively improves the functional properties of SPI by altering its conformation and structure. This study highlights the potential of ultrasound-assisted L-His modification in enhancing the functional properties of SPI for food applications.This study investigates the effects of ultrasound-assisted L-histidine (L-His) modification on the physicochemical properties and conformation of soybean protein isolate (SPI). The results show that ultrasound treatment reduces the particle size, increases solubility, and improves the homogeneity of SPI. The addition of L-His further reduces particle size, increases zeta potential, and enhances solubility and functional properties. Spectral analysis indicates that L-His forms a complex with SPI, altering the microenvironment of amino acid residues and improving emulsification and antioxidant properties. At a concentration of 0.3% w/w, the nanocomplex exhibits a smaller particle size (140.03 nm), higher zeta potential (-23.63 mV), and higher emulsification stability (22.48 min). The study also reveals that ultrasound and L-His treatment alter the secondary structure of SPI, reducing alpha-helix content and changing the microenvironment of hydrophobic amino acids. The optimal emulsification and antioxidant properties of SPI were achieved at a L-His concentration of 0.3%. The combination of ultrasound and L-His treatment effectively improves the functional properties of SPI by altering its conformation and structure. This study highlights the potential of ultrasound-assisted L-His modification in enhancing the functional properties of SPI for food applications.