The method of indirect competitive ELISA, scanning electron microscopy (SEM) and dynamic light scattering (DLS) were used to study the ultrasound-assisted glycation with lactose (UAG) on changes in allergenicity and structure of β-lactoglobulin (β-Lg) during digestion in vitro. The results showed that the allergenicity and the degree of hydrolysis of UAG modified β-Lg had a reduced during gastrointestinal digestion. Glycation limited β-Lg to form a smaller particle structure during gastric digestion, then aggregated during intestinal digestion. The fluorescence intensity first increased and then decreased, and the maximum emission wavelength appeared red shift. These results indicated that UAG modification could effectively reduce the allergenicity of protein during digestion. The decrease of allergenici-ty was closely related to structural changes of UAG modification.