Tofu prepared by conventional methods often has a bitter taste and poor water-holding capacity (WHC). To improve the quality of the product, alternative processes must be developed. Herein, the effect of ultrasound pretreatment on the properties of soymilk and tofu gel derived thereof were investigated. Treatment of soymilk with ultrasound gave rise to a reduction in the particle size and an enhancement in the surface hydrophobicity, whereby optimum values were obtained after 15 min treatment. Subsequently, microbial transglutaminase (MTG) was added to ultrasound-treated soymilk to promote the soy protein crosslinking. The gel strength, WHC, and nonfreezable water content of MTG-catalyzed tofu gel obtained from treated soymilk increased with the extension of the ultrasound pretreatment time, whereas the free sulfhydryl content decreased because of the formation of disulfide bonds. Fourier transform infrared spectroscopy demonstrated variations in the secondary structure of MTG-catalyzed tofu gel. Furthermore, soymilk's exposure to high-intensity ultrasound pretreatment led to a tofu gel with a dense, homogenous, and stable network structure, as evidenced by scanning electron microscopy. Therefore, this study answers for the theoretical support of the industrial production of MTG-catalyzed tofu gel from ultrasound-treated soymilk. PRACTICAL APPLICATION: High-intensity ultrasound pretreatment improved the texture properties of MTG-catalyzed tofu gel. The resulting MTG-catalyzed tofu gel has potential application in industrial production.