In this study, the infectious hematopoietic necrosis virus (IHNV) isolate SD-12 glycoprotein (glycoprotein, G) gene was cloned into commercial vector pcDNA3.1 (+) to construct an IHNV G expression vector, known as an infectious hematopoietic necrosis (IHN) nucleic acid vaccine and named pIHNsd-G. Rainbow trout (5±0.5) g were immunized with 2 μg of the vaccine by intramuscular injection at the dorsal fin base. The expression of the gene was detected by real-time PCR in the anterior kidney and the muscles from vaccine-delivered sites of the rainbow trout at 4 days and 7 days after immunization, respectively. At 21 days post immunization, the relative protection rate (RPS) of the vaccine was calculated by challenge with SD-12 at a dose of 100% of the tissue culture-infective dose (TCID₅₀) by intraperitoneal injection. The serum neutralizing antibody titers of immunized rainbow trout were tested at 60 and 150 days post-immunization. Finally, the pIHNsd-G promoter sequence and ampicillin resistance gene sequence were used as target genes and the dynamic distribution of pIHNsd-G in the inoculated part of the rainbow trout was monitored by PCR. The results showed that the gene was significantly up-regulated in the kidney and the muscles at the inoculated site and was significantly higher in the muscle tissue than in the head kidney at the same time point. Challenge of rainbow trout showed that the RPS of pIHNsd-G for rainbow trout was 94.4%. The neutralizing antibody titer revealed that at 60 days post-immunization, neutralizing antibodies were found in all of the serum samples of immunized rainbow trout and the highest titer was 320, while the highest antibody titer at 150 days post-immunization was 80. Since then, it has been shown that an effective IHN nucleic acid vaccine has been successfully obtained. The results of PCR following the injection of pIHNsd-G rainbow trout showed that all pIHNsd-G target fragments were detected in the muscle of the injection site on the first day after immunization; it was not possible to amplify the full-length ampicillin resistance gene from the injection site muscle at 84 days, and all target genes disappeared at 150 days. Based on the successful construction of an IHN nucleic acid vaccine and the systematic validation of its effectiveness, this study carried out dynamic analysis of the vaccine at the inoculation site, which provides basic data for the research and development of IHN nucleic acid vaccines and safety evaluations.