A pathogenic bacterium (S636), identified as Streptococcus iniae, was isolated from turbot (Scophthalmus maximus) in 2005. We immunized turbot with formalin-killed S. iniae four times (on days 1, 14, 21, and 28) by intraperitoneal inoculation. After each vaccination, we obtained serum samples and isolated the lymphocytes from the peripheral blood, spleen, pronephros, and mesonephros. We measured surface Ig-positive (sIg+) lymphocytes and serum antibody levels from these organs using flow cytometry and enzyme-linked immunosorbent assay (ELISA), respectively, using monoclonal antibodies against turbot immunoglobulin. We confirmed that the antibody reacted with both the surface and plasma Ig by confocal laser scanning microscopy and electron microscopy. The percentage of sIg+ in the lymphocytes increased following each successive vaccination. The mean percentage increased from 31.96% (control) to 37.49%, 38.36%, 42.9%, and 51.63% in the peripheral blood; from 27.09% to 36.63%, 36.81%, 39.28%, and 46.0% in the spleen; from 22.2% to 28.99%, 29.21%, 32.83%, and 41.58% in pronephros; and from 18.12% to 22.17%, 22.45%, 25.69%, and 31.68% in the mesonephros. The ELISA results were consistent with these results. Both the total and specific antibody levels increased with each vaccination. The mean OD value of the specific antibody assay increased from 0.094, to 0.269, 0.283, 0.333, and 0.421; for total antibody the mean OD value increased from 0.133, to 0.292, 0.323, 0.413, and 0.527.