Cyprinid herpesvirus 3 (CyHV-3), also known as koi herpesvirus (KHV), is a highly contagious and lethal virus. At present, a kind of commercial attenuated vaccine has been used in Israel against this virus. However, this vaccine, as well as other reported gene-modified attenuated vaccines against CyHV-3 infection, cannot exclude the risk of reversion to virulence. In contrast to live vaccines, DNA vaccines are easy to produce and safe to apply. CyHV-3 has a genome of 295 kb, which is predicted to encode 164 different open reading frames (ORFs). Among them, ORF148 encodes a glycosylated type I membrane protein and bioinformatics analysis has shown that this protein has good immunogenicity. During our previous study, CyHV-3 ORF148 was inserted into pEGFP-N1, generating a pEGFP-ORF148 recombinant plasmid. In the present study, it was confirmed that a pORF148-EGFP fusion protein could be successfully expressed by transfection assay and immunofluorescence analysis, respectively. The carps, n=45), were intramuscularly injected with the pEGFP-ORF148 recombinant plasmid, pEGFP-N1 and PBS three times at two-week intervals. The results of ELISA showed that immunization with the pEGFP-ORF148 recombinant plasmid could significantly (<0.01) improve the tire of specific antibodies against CyHV-3. The RT-qPCR revealed that injection with the pEGFP-ORF148 recombinant plasmid caused 145.30-, 131-, 52.27-, 12.16-, 4.19-, 3.05-, 4.03-fold increases of , gene expression in the head kidney, respectively, when the PBS immunized carps served as a control group. In the spleen, the expression of from pEGFP-ORF148 recombinant plasmid injected carps were 22.44-, 4.63-, 6.14-, and 4.23 times higher than those of control group. Post-infection with CyHV-3, the survival rates of carps immunized with PBS, pEGFP-N1, and the pEGFP-ORF148 recombinant plasmid were 30%, 35% and 85%, respectively, suggesting that immunization with the pEGFP-ORF148 recombinant plasmid significantly protected the carps against the CyHV-3 infection (<0.01). In conclusion, DNA vaccination based on the pEGFP-ORF148 recombinant plasmid could elicit the activation of an immune response and production of a specific antibody to produce effective protection against CyHV-3 infection. Thus, this study highlights a potential DNA vaccine candidate against CyHV-3.