T and B lymphocytes are closely related to immunization and pathogen infection. Our previous study confirmed the CD3+, CD4-1+, CD4-2+, CD8β+ T lymphocytes and IgM+ B lymphocytes presented in the peripheral blood leukocytes (PBLs) of flounder (Paralichthys olivaceus), in this paper, the variations of T and B lymphocytes of flounder after Hirame novirhabdovirus (HIRRV) infection or immunization were investigated. The flounders were injected with live or inactivated HIRRV, then the percentages of T and B lymphocytes in PBLs were analyzed by Flow cytometry (FCM), total antibodies and HIRRV-specific antibodies in serum were detected by Enzyme-linked immunosorbent assay (ELISA), and expression of twelve immune-related genes in the head kidneys were determined using q-PCR. The results showed that the percentages of CD3+, CD4-1+, CD4-2+, CD8β+ T lymphocytes and IgM+ B lymphocytes significantly increased in both infection and immunization groups, in infection group they decreased rapidly after the peak and significantly lower than control levels at the end of infection, in immunization group they went down steadily to the control levels at the end of immunization. The total antibodies and HIRRV-specific antibodies increased first and peaked on the 7th day post infection and on the 14th day post immunization, respectively, then gradually decreased to the control levels. Additionally, twelve immune-related genes were up-regulated in both groups. These results demonstrated that the HIRRV induced both humoral and cellular immunity of flounder, the lymphocytes varied more sharply in infection group than those in immunization group and CD8+ T lymphocytes responded much more than CD4+ T lymphocytes to HIRRV antigen.