Takifugu obscurus is a farmed fish of great economic importance in China. The rapid development of T. obscurus aquaculture industry has been accompanied by disease and low-temperature stress, resulting in huge economic losses. Cell lines are used extensively in teleost physiology and pathology as the most cost-effective platform for in vitro research. A novel gill cell line of T. obscurus (named TOG) was first successfully established, and passed through 52 generations. The optimal conditions for TOG growth were 20 % FBS concentration and 24 °C, TOG could be grown in both hypotonic (150 mOsmol-kg-1) and hypertonic (600 mOsmol-kg-1) environments. TOG was determined to be derived from T. obscurus by sequencing the mitochondrial COI gene. Karyotype analysis revealed that the chromosome number of TOG was 44 (2n = 44). Transfection experiment showed that TOG was able to express foreign genes. Furthermore, several immune-related genes were significantly up-regulated in TOG after LPS and poly (I:C) stimulation, including tlr3, isg15, il1β and il10. Additionally, transcriptome analysis of TOG under low-temperature stress (24 °C, 18 °C, 12 °C, 10 °C and 8 °C) found that differentially expressed genes (DEGs) were significantly clustered in several immunological and energy metabolic pathways, and cold stress could disrupt the immune barrier and reduce immunity by downregulating the immune-related pathways. Additionally, weighted gene co-expression network analysis (WGCNA) revealed that bule module and turquoise module, which were closely correlated with low temperature and the degree of fish damage, were both predominantly found in PPAR, NOD-like receptor and Toll-like receptor signaling pathway. Hub genes were identified in these two modules, including mre11, clpb, dhx15, ddx18 and utp15. TOG cell line will become an effective experimental platform for genetic and immunological research, and our results would help us gain a deeper insight into the molecular mechanism of cold tolerance in teleost.