The efficacy of the alginate microsphere (Alginate MS) oral vaccine against Miamiensis avidus in olive flounder (Paralichthys olivaceus) was confirmed through challenge infections by both im- mersion and injection routes. In trial 1, the formalin-inactivated M. avidus coated with alginate, des- ignated as ‘IMa+Alginate MS’ group, and the IMa group were administered with vaccines mixed with feed, with a total antigen dose of 3.75 × 106 cells/fish. When challenged with immersion infection at five weeks post vaccination, the relative percent survival (RPS) in the IMa+Alginate MS group was 50% (immersed in 50% seawater) and 37.5% (immersed in 100% seawater). The group that received only IMa showed a low survival rate. In trial 2, the antigen was fed mixed with feed at a total dose of 2.38 × 106 cells/fish for 5 days. Two weeks after oral vaccination, fish were intra- peritoneally injected for infection. The RPS in the IMa+Alginate MS group was 30.8%, while the IMa-only group showed no vaccine efficacy. At five weeks post vaccination, when subjected to chal- lenge infection by immersion in 50% seawater, the IMa+Alginate MS group recorded a RPS of 42.9%, whereas the IMa group had a RPS of 14.3%. The results of this study indicate that coating M. avidus antigen with alginate can provide...