Abstract: In order to develop a DNA vaccine against Cyprinid herpes virus 3 (CyHV-3), also known as Koi herpesvirus (KHV), we inserted CyHV-3 ORF65  into pEGFP-N1 to construct recombinant plasmid pEGFP-ORF65. The expression of pORF65-EGFP fusion protein in Cyprinus carpio var. Jian was confirmed by transfection and in vivo imaging experiments. The recombinant plasmid was injected into the tail stalk of the carp seedlings at dosage of 3 μg·tail⁻¹ for about 10 g and immunized for three times. ELISA detection shows that pEGFP-ORF65 could significantly increase the level of serum specific antibodies. The challenge test shows that 21 d after CyHV-3 challenge, the survival rates of PBS group, pEGFP-N1 group and pEGFP-ORF65 group were 25%, 30% and 90%, respectively, which significantly improved the survival rate of the carp seedlings (P<0.01). The study provides a basis for the application of ORF65 as a target gene of DNA vaccine in the prevention and control of CyHV-3.