2'-Fucosyllactose (2'-FL), an industrial breast milk oligosaccharide, is approved for use in infant formula and may reduce cow's milk protein allergenicity. To investigate whether glycosylation products of 2'-FL in dairy products (2'-FL-β-LG) increase its sensitization, we cross-linked β-LG with 2'-FL and used it to sensitize Balb/c mice, comparing it with nonglycosylated β-LG. Both 2'-FL-β-LG sensitization and oral 2'-FL intervention reduced allergic symptoms, specific antibodies (IgE, IgG, and IgG2a), inflammatory cytokine levels, and intestinal damage. 2'-FL also shifted T-cell differentiation, reduced cell surface expression of DC receptors, and enhanced gut microbial diversity. Oral 2'-FL showed the greatest efficacy, suggesting its potential for lowering milk allergenicity in formula.