ABSTRACT IFN-γ (Interferon-gamma), Mx and IRF-1 (Interferon regulatory factor-1) are main immune-related genes and they play important roles in the innate immune system of vertebrates. In this study, the expression level of the three immune-related genes in twelve tissues of normal adult Japanese flounder was detected using semi-quantitative RT-PCR. Thirteen time points (3h, 6h, 9h, 12h, 24h, 36h, 48h, 60h, 72h, 84h, 96h, 108h, 120h) were selected to analyze the expression of IFN-γ, Mx and IRF-1 in spleen, head kidney, liver of Japanese flounder infected with Lipopolysaccharide (LPS). The Japanese flounder IFN-γ, Mx and IRF-1 genes were differently expressed in these tissues and had high expression levels in classical fish immune organs like spleen and head kidney. It was found that the highest expression levels of the Japanese flounder IFN-γ were detected at 24h in spleen, 36h in head kidney and 48h in liver after challenge with LPS. Interestingly, the highest expression levels of Mx in spleen and head kidney were both at 36h and IRF-1 in spleen and liver were both at 24h. The highest expression level of Mx in liver was at 48h and IRF-1 in head kidney was at 12h. The study provides a basis for further research on immune mechanism of IFN-γ, Mx, IRF-1 and the production of recombinant IFN-γ, Mx or IRF-1 used in Japanese flounder cultivation in future.