) on TOF proliferation showed that the highest multiplication rate of TOF was obtained at 6‰ NaCl mass and 4 g/L alkalinity, respectively. Furthermore, the expressions of several genes associated with the immune response were significantly upregulated after stimulating TOF cells with lipopolysaccharide (LPS) and poly (I:C), including irf7, il10, myd88, and nod1. Additionally, the Aeromonas hydrophila infection assay with TOF cells showed that TOF cells were sensitive to bacteria, and pufferfish could promote antimicrobial activity through NLR/NF-κB pathway to regulate the production of cytokines. This study suggested that TOF cell line could be an advantageous in vitro model for the investigation of fish immunology and virology, and could assist us to better comprehend the T. obscurus immune response to bacterial invasion.