β-Lactoglobulin (BLG) is a main allergen in cow’s milk, which can cause severe allergic reactions. This study aimed to evaluate the effect of co-modification with ferulic acid (FA) and docosahexaenoic acid (DHA) on the antigenicity and structural properties of BLG. A BLG-DHA-FA complex was obtained by non-covalent binding and its structural characteristics and antigenicity were investigated by various spectroscopic techniques, molecular docking and indirect competitive enzyme-linked immunosorbent assay. The results showed that the combination of FA and BLG-DHA changed the microenvironment surrounding aromatic amino acids, enhancing the polarity. FA and DHA bound to the hydrophobic cavity of BLG through hydrophobic interaction and hydrogen bonding, thereby changing the secondary structure of BLG. Specifically, the contents of β-sheets and random coils were reduced, and the β-turn content was increased. Compared with BLG and BLG-DHA, the addition of FA significantly reduced the antigenicity, and the highest inhibition rate against the immunoglobulin G (IgG) binding ability of 40% was obtained when the FA concentration was 0.08 mg/mL.