Abstract: A method for extracting and separating high-purity tetrodotoxin (TTX) from pufferfish viscera, comprising the following steps: adding an acid alcohol homogenate to pufferfish viscera, adding a certain amount of a flow aid and stirring uniformly, loading into a chromatographic column, eluting with the same acidic alcohol, collecting an eluent containing TTX, and concentrating under reduced pressure to obtain a TTX concentrated solution; further separating using ion exchange resin column chromatography, collecting an eluent containing TTX, and using a nanofiltration membrane to perform concentration or reduced pressure evaporation to dryness to obtain a TTX crude product; and precipitating and crystallising the TTX crude product by means of a solvent to obtain TTX with a purity greater than 98%.