Abstract The fish immune system is comprised of the non-specific and specific immune systems, having both humoral and cellular mechanisms to resist against infectious diseases. However, the biodefense- and immune-related systems of fish are not clearly understood at the molecular level. Characterization of the immune system of fish is important for the development of vaccines and other infectious disease prevention methods. In view of the above, we conducted an expressed sequence tags (ESTs) analysis of Japanese flounder, Paralichthys olivaceus for characterization of its immune system at the molecular level. We sequenced more than 7000 clones of 11 cDNA libraries. Numerous putative immune relevant genes, such as cytokines, cytokine receptors, cell surface molecules, transcription factors, complement component, anti-microbial proteins, proteases and protease inhibitors were identified. We also cloned and characterized several immune relevant genes from the BAC library. Currently the expression pattern for most of immune- and defense-related genes, from LPS or ConA/PMA treated peripheral blood leukocytes, after DNA vaccination study and after viral infection were characterized by microarray spotted with 1200 different Japanese flounder cDNAs. The expression patterns of these genes were varied in many of the genes examined, in different stimulations and time points. For the functional study of Japanese flounder's immune-relevant genes, we made recombinant proteins in insect cell system and characterized their activities. This review focuses on toll-like receptors, lysozymes, and T cell receptors of Japanese flounder and assessment of DNA vaccine using cDNA microarray of Japanese flounder.