Orf is an infectious disease caused by orf virus (ORFV). Animals can be infected by ORFV through contact and it is preferred to epithelial infection. The ORFV 020 gene is an important enzyme regulating protein activity of the virus. In this study, specific PCR primers were designed according to the sequence of the Orf 020 gene in GenBank. After the viral DNA was extracted from ORFV infected clinical samples, The Orf 020 gene was amplified by PCR and subcloned to pET-32a prokaryotic expression vector. The recombinant plasmid was identified and named as pET-32a-ORF-020, which was then transformed into E.coli competent cell BL21 and induced by IPTG. The expressed proteins were identified by SDS-PAGE and Western blot. The ORF 020 protein was then purified and immunized to BALB/c mice for antiserum preparation. The results of SDS-PAGE showed that the Orf 020 gene was correctly expressed in vitro. The protein size was about 37 kDa. The western-blot analysis showed that the prepared antiserum reacted specifically with the ORF 020 protein.