Aiming to study the effect of ultrasonic combined with enzyme treatment on the structure of soybean protein isolate (SPI) and the properties of SPI gel cross-linked with glutamine transaminase (TG). The effects of ultrasonic combined with enzyme treatment on the structure of SPI were analyzed by fluorescence spectroscopy and Fourier transform infrared spectroscopy. Meanwhile, the relationship between SPI stuctural changes and functional characteristics were studied by particle size, free sulfhydryl group, surface hydrophobicity analysis, texture, water holding analysis and scanning electron microscopy. The results showed that after ultrasonic treatment, papain treatment and ultrasonic combined with enzyme treatment, the proportions of the secondary structure fractions αhelix and βturn were decreased significantly (P<0.05), and the proportions of βsheet and random coil were increased significantly (P<0.05), the protein structure was extended, the free sulfhydryl group and hydrophobic group were exposed, thus the free sulfhydryl group content and surface hydrophobicity of SPI were increased. Compared with untreated SPI, the SPI gel prepared by ultrasound combined with enzyme treatment formed a uniform and dense gel network, and its gel strength and water holding capacity (WHC) were significantly improved, which were increased by (278.04±18.81)% and (89.51±2.78)%, respectively. In addition, the gel strength and WHC were higher in the SPI gel treated with a combination of ultrasound and enzyme than that treated with ultrasound or enzyme alone, exhibiting the synergistic effect of enzymatic and ultrasonic treatment. In summary, ultrasound combined with enzyme treatment can improve the structure of SPI and the characteristics of TG cross-linked SPI gel.