Dendritic cells (DCs) play a pivotal role in activating naïve T-cells and bridging innate and adaptive immunity. This study aimed to identify and characterize CD83 and CD276 as potential markers for DCs in olive flounder (Paralichthys olivaceus). Specific antibodies against these markers were developed and used to analyze their distribution in peripheral blood leukocytes (PBLs) and intestinal tissues. Flow cytometry and immunohistochemistry revealed that CD83 and CD276 are expressed on DCs, with peak expression observed one week after oral administration of an inactivated viral hemorrhagic septicemia virus (VHSV) vaccine. Gene expression analysis further demonstrated significant activation of immune-related genes, including CD3, IgM, and TLRs, indicating that oral vaccine administration effectively activates the intestinal mucosal immune system. These findings provide valuable insights into fish immune responses and establish CD83 and CD276 as promising DC markers, contributing to the development of mucosal vaccine strategies in aquaculture.