Fermentation techniques may induce alterations in fish allergen immunoreactivity. In this study, the influence of fermentation with three different strains of Lactobacillus helveticus (Lh187926, Lh191404, and Lh187926) on the immunoreactivity of Atlantic cod allergens was investigated via several methods. Gradually reduced protein composition and band intensity due to the fermentation by strain Lh191404 were found in SDS-PAGE analysis, and decreased immunoreactivity of fish allergens was confirmed by Western blotting and ELISA analysis due to the fermentation of strain Lh191404. Additionally, results from nLC-MS/MS and immunoinformatics tools analysis demonstrated that the protein polypeptide and allergen composition of Atlantic cod showed evident alterations after fermentation, with the epitopes of the main fish allergens being heavily exposed and destroyed. These results indicated that the fermentation of L. helveticus Lh191404 could destroy the structure and linear epitopes of the allergens from Atlantic cod and may have considerable potential in mitigating the allergenicity of fish.