<p>Viral hemorrhagic septicemia virus (VHSV) is a single-stranded RNA virus that infects both freshwater and marine fish. In Korea, VHSV genotype Ⅳa infects cultured olive flounder (<i>Paralichthys olivaceus</i>) annually. As a prevention strategy against infectious viral diseases, this study aimed to develop a multi-epitope DNA vaccine using <i>in silico</i> methods and evaluate its efficacy <i>in vivo</i> in olive flounder. Cytotoxic T-lymphocyte (CTL), helper T-lymphocyte (HTL), and linear B-lymphocyte (LBL) epitopes of the VHSV glycoprotein were predicted using bioinformatic tools and assembled with the linkers AAY, GPGPG, and KK, respectively. This multi-epitope sequence (359 bp) was then cloned into the pcDNA3.1(+) vector for use as a DNA vaccine, and this construct was named "pMulti". To evaluate the efficacy of the multi-epitope DNA vaccine, olive flounder were vaccinated with PBS, pcDNA3.1(+), or pMulti. The persistence and transcript levels were confirmed up to 28 days post-vaccination (dpv). Moreover, HTL-associated immune-related genes, including MHCII and IFN-γ, were significantly upregulated at 3 dpv. Immunoglobulin M (IgM) production was confirmed by ELISA at 14, 21, and 28 dpv. The protective efficacy of the vaccine was tested by challenging fish with 1×10⁴ PFU of VHSV at 21 dpv, and the relative percent survival (RPS) of pMulti-vaccinated fish was 40% in experiment 1 and 23.08% in experiment 2. Moreover, the hazard ratio for the pMulti vaccine was significantly lower than that for PBS and pcDNA3.1(+) (2.08 and 2.65, respectively). Although these findings demonstrate the potential of multi-epitope-based vaccines against VHSV IVa, additional research involving <i>in vivo</i> evaluation of a subunit vaccine through protein expression is essential for a more conclusive assessment of efficacy.</p>