In this study, we investigated the effects of different high-intensity ultrasound (HIU) pretreatment times (0–60 min) on the structure of β-conglycinin (7S) and the structural and functional properties of 7S gels induced by transglutaminase (TGase). Analysis of 7S conformation revealed that 30 min HIU pretreatment significantly induced the unfolding of the 7S structure, with the smallest particle size (97.59 nm), the highest surface hydrophobicity (51.42), and the lowering and raising of the content of the α-helix and β-sheet, respectively. Gel solubility showed that HIU facilitated the formation of ε-(γ-glutamyl)lysine isopeptide bonds, which maintain the stability and integrity of the gel network. The SEM revealed that the three-dimensional network structure of the gel at 30 min exhibited filamentous and homogeneous properties. Among them, the gel strength and water-holding capacity were approximately 1.54 and 1.23 times higher than those of the untreated 7S gels, respectively. The 7S gel obtained the highest thermal denaturation temperature (89.39 °C), G′, and G″, and the lowest tan δ. Correlation analysis demonstrated that the gel functional properties were negatively correlated with particle size and the α-helix, while positively with Ho and β-sheet. By contrast, gels without sonication or with excessive pretreatment showed a large pore size and inhomogeneous gel network, and poor properties. These results will provide a theoretical basis for the optimization of HIU pretreatment conditions during TGase-induced 7S gel formation, to improve gelling properties.