To extract tetrodotoxin with immunoaffinity cartridge, develop an ultra fast liquid chromatography coupled with triple quadrupole mass spectrometry (LC-MS/MS) method for determination of tetrodotoxin in fish muscle and liver, and provide proof for detection TTX in aquatic products.Methods Tetrodotoxin was extracted by 1% acetic acid methanol, supernatant added with PBS buffer was loaded on immunoaffinity cartridge, the eluent was blow-dried by nitrogen and dissolved in constant volume. Then the sample was separated on a Zic-Hilic column with 10 mmol/L ammonium formate-0.1% formic acid and acetonitrile as mobile phase by gradient elution. Detection was carried out by multiple reaction monitoring. Results Tetrodotoxin had a good linear result in the range of 1.0-1 000.0 ng/ml with detection limit of 0.3 μg/kg in muscle and 0.2 μg/kg in liver respectively. The recovery was 52.4%-72.6%.Conclusion This method has excellent specificity, good effect of extraction and without matrix inhibition effect. The assay is suitable for trace detection of tetrodotoxin in fish muscle and liver.