Hematological analysis can provide crucial information for monitoring the health of fish. However, there is no current information available regarding hematological changes in olive flounder following infection by Vibrio scophthalmi. In this study, hematological and biochemical alterations were determined in olive flounder infected by the high virulence strain (HVS) and low virulence strain (LVS) of V. scophthalmi. Survival in serum, skin mucus, and macrophages of olive flounder was also compared between the HVS and LVS. The results demonstrated that the hematocrit value in infected fish declined from 23.4% at 0 h to 18.0% at 168 h post infection. The total protein concentration in fish infected with the HVS was significantly higher than in fish infected with the LVS and a non-infected control. Lysozyme activity was significantly different between infected and control fish. The HVS survived in serum and cell numbers increased substantially, while cell numbers of the LVS in serum decreased. These changes in hematological characteristics in fish infected by V. scophthalmi can be used as an effective and sensitive index to monitor the physiological and pathological conditions of fish. The survival and reproduction of V. scophthalmi in host serum, skin mucus, and macrophages play a major role in systemic infection and can serve as a virulence indicator for different strains.