A new cell line was established from the brain of a cultured fish, tilapia (Oreochromis niloticus), designated as TA‐02 (Tilapia Astrocyte clone 02 cell line). The TA‐02 cells are grown for 300 days in an L‐15 medium supplemented with 10% fetal bovine serum (FBS). This cell line showed excellent proliferative capacity and expressed various neuroglial cell markers, including SOX2, SOX10, Hes1, Notch1, Occludin, E‐cadherin, and GFAP. In addition, TA‐02 cells were susceptible to Tilapia Lake Virus (TiLV) as demonstrated by the presence of a severe cytopathic effect (CPE), virus particle in a transmission electron microscope (TEM), and PCR positive signal. Bacterial cytotoxicity studies showed that Streptococcus agalactiae was toxic to TA‐02 cells. When co‐culture with trans‐well, TA‐02 exhibited prominent barrier properties, manifested by tight intercellular junctions and increased trans‐endothelial electrical resistance (TEER). In addition, the barrier is effective against Escherichia coli (non‐meningitis pathogenic bacteria). In contrast, S. agalactiae (meningitis pathogenic bacteria) can pass through the membrane comprising the cells in the trans‐well insert. The newly established TA‐02 cell line provided a valuable tool for virus pathogenesis and a vitro model of the fish blood–brain barrier.