Bovine α-lactalbumin (BLA) induced severe cow’s milk allergy. In this study, a novel strategy combining ultrasonication, performed before glycation, and phosphorylation was proposed to reduce BLA allergenicity. Result showed that IgE- and IgG-binding capacities and the release rates of histamine and interleukin-6 from RBL-2H3 were reduced. Moreover, intrinsic fluorescence intensity and surface hydrophobicity were decreased, whereas glycated sites (R10, N44, K79, K108, N102 and K114) and phosphorylated sites (Y36 and S112) of BLA were increased. Minimum allergenicity was detected during BLA treatment after ultrasonic prior to glycation and subsequent phosphorylation because of considerable increase in glycated and phosphorylated sites. Therefore, the decrease in allergenicity of BLA, the effect correlated well with the shielding effect of glycated sites combined with phosphorylated sites and the conformational changes. This study provides important theoretical foundations for improving and using the ultrasonic technology combined with protein modification in allergenic protein processing.