Abstract: Tetrodotoxin (TTX) has an excellent application prospect in the treatment of neuropathic diseases such as sedation, analgesia, anesthesia and detoxification without causing addiction. However, due to its high cost of extraction from puffer fish, it is difficult to be widely applied. There is increasingly evidence that TTX originates from its symbiotic microbes. A strain of TTX-producing Bacillus sp. 3G2 was isolated from the liver of wild puffer fish (Takifugu oblongus), and 35 ng/mL TTX in fermentation broth was detected by ELISA and colloidal gold assay. Then, Bacillus sp. 3G2 was inoculated in a special dialysis incubator, and continuous culture was achieved by continuous flow and fresh culture medium. The mice biological activity and tetrodotoxin test strips were used to detect TTX in the fermentation broth. The crude TTX was purified by TTX antibody immunoprecipitation and further identified by high performance liquid chromatography (HPLC). Our study further corroborated the exogenous hypothesis of tetrodotoxin by means of screening highquality strains from puffer fish, and preliminarily verify the superiority of dialysis production of tetrodotoxin. Therefor it provided a practical basis for subsequent commercial application.