Abstract: 【Objective】This study aimed to express the NS5A protein of Bovine Viral Diarrhea Virus (BVDV) using a prokaryotic expression system and to prepare polyclonal antibodies against it, thereby providing technical support for the prevention and control of BVDV.【Methods】The physicochemical properties of the BVDV NS5A protein were analyzed using bioinformatics software. The recombinant plasmid pET-28a-NS5A was constructed and transformed into competent E. coli BL21 cells. The NS5A protein was induced for expression, followed by urea denaturation and purification via Ni2?-NTA affinity chromatography. The purified recombinant protein was emulsified with adjuvant and used to immunize BALB/c mice multiple times to generate polyclonal antibodies. Antibody titers were determined by indirect ELISA, and the antibodies were further characterized by indirect immunofluorescence assay (IFA) and Western blot. 【Results】Bioinformatics analysis indicated that the NS5A protein lacks a signal peptide and transmembrane domains and is hydrophilic. The prokaryotic expression plasmid pET-28a-NS5A was successfully constructed, and the recombinant NS5A protein with an approximate molecular weight of 58 kDa was expressed. After urea denaturation and Ni2?-NTA affinity chromatography, a high-purity recombinant NS5A protein was obtained. Indirect ELISA showed that the antibody titer exceeded 1:409,600. Both Western blot and IFA results demonstrated that the polyclonal antibodies exhibited good reactivity and specificity. 【Conclusion】These results indicate the successful preparation of highly specific polyclonal antibodies against NS5A, which provides a foundation for basic research and clinical diagnosis of BVDV.