Objective To clone, express, purify a fragment of β-lactoglobulin, and to identify its allergenicity. Methods RT-PCR method was applied to clone the cDNA of a fragment of β-lactoglobulin, and then the fragment cDNA was sequenced and sub-cloned into pET expression vector. The cloned gene was expressed in E. coli origami induced by IPTG. The recombinant protein was purified by metal (Ni²⁺) chelating affinity chromatography. The allergenicity was examined by both Western blotting and enzyme-linked immunosorbent assay (ELISA). Results The recombinant fragment of β-lactoglobulin gene was cloned with a 264 bp open reading frame coding for 87 amino acids. The fragment of β-lactoglobulin was expressed both as inclusion bodies and soluble protein. The soluble protein was purified, and the immunogenicity of the protein identified by ELISA was good. Conclusion The clone and expression of the fragment of β-lactoglobulin was successful; which would provide references for the following studies on testing the immunogenicity of milk and lay great foundations for developing monoclonal antibodies and detection kits for the major allergens of milk.