Abstract: We amplified Koi herpesvirus ORF59 gene by PCR using template DNA extracted from the kidney of Cyprinus carpio koi infected with Koi herpesvirus (KHV). The full length of the gene is 411 bp encoding an envelope protein which consists of 136 amino acids. The predicted molecular weight of KHV ORF59 is 14.3 kDa and its estimated isoelectric point is 6.91. Altogether 12 potential O-glycosylation sites are found in this protein. There is a mutation from G to A at 130th site of cloned KHV ORF59 gene and it causes an amino acids substitution from Ala to Thr. With the software DNA Star and based on the analysis of the flexibility in second structure , hydrophilicity, surface probability and antigenic index of KHV ORF59 protein, the B cell epitopes are predicted. The sequence encoding the major epitope domain and the complete coding sequence of KHV ORF59 gene were subcloned into pET-32a(+) vector to construct recombinant plasmids which named pET32a-ORF59S and pET32a-ORF59C, which then were transformed into E.coli Rosetta respectively and expressed by IPTG inducement. SDS-PAGE and Western blot results show that pET32a-ORF59S can be highly expressed and the truncated KHV ORF59 protein is mainly expressed as soluble. With His Bind Resin filling, the truncated protein is purified.