<p>Single-cycle viruses generated by the deletion of gene(s) essential for viral replication in the genome can be a way to enhance the safety of attenuated virus-based vaccines because single-cycle viruses can infect cells only once and cannot produce infective viral particles. In the present study, not only to guarantee safety as a prophylactic vaccine but also to enhance the protective efficacy, a single-cycle viral hemorrhagic septicemia virus (VHSV) lacking the G gene and containing olive flounder (<i>Paralichthys olivaceus</i>) interferon-γ (IFNγ) ORF between N and P genes of the viral genome (rVHSV-A-IFNγ-ΔG) was rescued using reverse genetic technology, and its protective potential was evaluated through the immunization of olive flounder. In the challenge experiment, the cumulative mortality in the control group reached 80%, while groups of fish immunized with various doses of rVHSV-A-IFNγ-ΔG (ranging from 1 × 10³ to 1 × 10⁵ pfu/fish) showed no mortalities. In contrast, fish immunized with various doses of rVHSV-ΔG exhibited 0–20% mortality in a dose-dependent manner. Serum ELISA analysis revealed that groups receiving the highest doses of rVHSV-ΔG and rVHSV-A-IFNγ-ΔG showed significantly elevated titers compared to the control group. Although no statistically significant differences in ELISA titers were observed among groups immunized with lower doses of recombinant VHSVs, particularly at 1 × 10³ pfu/fish, and the control group, the markedly higher protection compared to the control group suggests that these single-cycle viruses possess a limited capacity to induce antibody responses, yet are capable of conferring protective immunity against VHSV independently of humoral mechanisms. Moreover, the complete protection achieved with low-dose immunization of rVHSV-A-IFNγ-ΔG indicates its strong potential as a highly effective prophylactic vaccine candidate.</p>