Cyprinid herpesvirus 2 (CyHV-2) is a highly contagious pathogen that causes haematopoietic necrosis disease in goldfish (Carassius auratus auratus) and gibel carp (Carassius auratus gibelio). Currently, there are no licensed vaccines or therapeutics for CyHV-2. Nonetheless, pVAX1 is a eukaryotic plasmid designed for the development of animal DNA vaccines. This vector allows high-copy number replication in E. coli and contains a CMV promotor for high-level expression of foreign proteins. In this study, the coding region of the CyHV-2 major capsid protein ORF66 was amplified by PCR and cloned into the pVAX1 vector to construct a recombinant plasmid, pVAX-ORF66. Additionally, a recombinant prokaryotic expression plasmid, pRSET-ORF66, was constructed and transformed into E. coli BL21(DE3)pLysS. The ORF66 recombinant protein was then produced by induction with 1 mmol/L IPTG; subsequently, the polyclonal antibody was prepared by subcutaneously immunizing New Zealand rabbits with the purified protein. The titer and specificity of the prepared antibody were analyzed using an ELISA assay, Western blotting, and an indirect immunofluorescence assay (IFA). Next, the recombinant plasmid pVAX-ORF66 was transfected into goldfish brain (GFB) cells, and the expression of ORF66 protein was detected by IFA and Western blotting. Finally, pVAX-ORF66 was intramuscularly injected into gibel carp at the anterior-to-dorsal fin region, before being infected with CyHV-2. The results demonstrated that the full nucleotide length of ORF66 was 1200 bp and the molecular weight of the recombinant protein was approximately 45 kDa, which were both consistent with their predicted sizes. The prepared rabbit polyclonal antibody was highly specific to CyHV-2, with a minimum titer of 1:20000. ORF66 expression could be detected in both infected and pVAX-ORF66 transfected GFB cells and was mainly distributed in the cytoplasm. Finally, the relative survival rate of immunized gibel carp was determined to 55.6%. Collectively, in this study, a eukaryotic expression plasmid, pVAX-ORF66, was constructed that could serve as a DNA vaccine for the prevention and control of CyHV-2. These results also provide a basis for further studies regarding the molecular function of ORF66 during CyHV-2 infection.