Site specific polyethylene glycol modification (PEGylation) catalyzed by transglutaminase (TGase) was applied to modify β-lactoglobulin (β-LG) at glutamine residues and its effect on the antigenicity of β-LG was explored. The modification rate of the modified product was analyzed by SDS-PAGE combined with gel filtration chromatography. According to single factor experiment, the optimal modification conditions were obtained as follows: reaction pH was 7.0, reaction temperature was 25 ℃, molar ratio of β-LG to PEG was 115, mass ratio of TGase to β-LG was 31, the amount of ethanol added in buffer was 25% (v/v). Under the optimal modification conditions, the modification rate of PEGylated product was 60.17%. The PEGylated product was purified by cationic exchange chromatography, the SDS-PAGE analysis result showed that the PEGylated product had an apparent molecular mass of about 28 kDa, which was mono-PEGylated product. The result of reversed phase high performance liquid chromatography showed that the obtained PEGylated product was homogeneous and without other isomers existed. The indirect competitive ELISA result showed that the antigenicity of β-LG was significantly reduced after PEGylation, and the antigenicity reduction rate was 59.04%.