Abstract: Cyprinid herpesvirus 3 (CyHV-3) envelope protein pORF65 has 1 785 bp DNA length encoding 594 amino acids. In this research, the codons of pORF65 were optimized by analysis of rare codon, prediction of signal peptide and transmembrane domains. Then the pET32a-trunORF65 plasmid was constructed, containing the pORF65 complete encoding sequence without N terminal signal peptide and C terminal transmembrane segment. Moreover, according to the analysis of DNAStar, ABCpred and BepiPred softwares, the sequence encoding the major B cell epitopes of pORF65 was amplified by SOE PCR and inserted into pET-32a (+), which was named as pET32a-modORF65. Then the pET32a-trunORF65 and pET32a-modORF65 plasmids were transformed separately in E.coil BL21 strain and induced by IPTG. The results of SDS-PAGE and Western blot analysis show that the pET32a-modORF65 plasmid can express the target protein efficiently and the molecular weight of recombinant protein was 56.4 kD. In addition, the koi serum IgM was purified through rProtein G and was used to prepare mouse anti-koi IgM polyclonal antibody. On this basis, indirect ELISA method was established using purified pORF65 as coating protein and mouse anti-koi IgM as detect antibody, which was used in the titer detection of koi serum collected from the koi immunized with pEGFP-ORF65 DNA vaccine.