Abstract The aim of this work was to establish and optimize the phosphorylation reaction conditions of parvalbumin (PV), the major allergen of silver carp (Hypophthalmichthys molitrix), and to determine the influence of phosphorylation reaction on the structural and immunological properties of PV. By mixing PV with D-Glucose-6-phosphate disodium salt hydrate (G6P), phosphorylation of PV was carried out by dry-heating treatment at different concentration, duration and temperatures. The results indicated that the optimal condition for phosphorylation reaction of PV was at 80 °C for 80 min with the ratio of PV to G6P as 1:4 (weight ratio). Dimers and polymers were formed in the phosphorylated sample, which might be attributed to the covalent bonds formed during phosphorylation reaction as analyzed by Tricine-SDS-PAGE. Formation of compact aggregates after phosphorylation reaction was further confirmed by scanning electron microscopy (SEM), which might block the access of immunoglobulins to the epitopes, and therefore decrease the antigenicity of G6P-PV, as demonstrated by dot-blotting using mouse anti-silver carp PV monoclonal antibody. Circular dichroism spectroscopy (CD) analysis revealed that phosphorylation reaction affected the secondary structures of PV significantly. Furthermore, surface hydrophobicity of PV was measured using hydrophobic fluorescence probes, 8-anilino-1-naphthalenesulfonate (ANS). The results revealed that the hydrophobicity of phosphorylated PV was enhanced, as the fluorescence intensity increased. The aggregation formation and change in the secondary structures of G6P-PV might explain the decrease of PV antigenicity after phosphorylation reaction. Our present work strongly suggested the effects of phosphorylation reaction on the decrease of antigenicity of food allergens.