Fish cell lines are important tools in many biological studies. Here, we established a new Japanese flounder brain (JFB) cell line from the brain tissue of the Japanese flounder (Paralichthys olivaceus), which was successfully sub-cultured for over 120 passages, and consisted predominantly of fibroblast-like cells. Sequencing of the actin gene confirmed that JFB cells were derived from the Japanese flounder, and JFB was also identified as a neural stem cell line on the basis of the mRNA expression of nestin gene. The JFB cells could grow at a temperature ranging from 17 to 29 °C, and 23 °C was the optimum temperature. Karyotype analysis showed that the chromosome mode number was 48, demonstrating a normal diploid chromosome number. The transfection efficiency of pEGFP-N1 in JFB cells was up to 30%. The JFB cells showed susceptibility to Hirame rhabdovirus (HIRRV), Lymphocystis disease virus (LCDV), Bohle virus (BIV) and Chinese giant salamander iridovirus (GSIV), which was demonstrated by varying degrees of cytopathic effects. Viral infection or Poly (I:C) stimulation upregulated the expression of multiple immune-related genes, including IL-1β, IL8, Mx and TNF. These results showed that the newly established JFB cell line is an ideal tool for studying gene manipulation, host-virus interaction and potential vaccines development.