To establish a method of multiple reaction monitoring technology (MRM) by high performance liquid chromatography tandem mass spectrometry (HPLC-MS/MS) to detect tetrodotoxin (TTX) content in fish.Methods Fish samples were ultrasonic extracted by 80% acidified methanol solution (containing 0.1% acetic acid), and purified by dichloromethane, centrifuged by ultrafiltration, and analyzed after membrane filtration. TTX was separated on a HILIC column, with acetonitrile-0.06% ammonium formate solution (V/V=65∶35, pH=3.5) as the mobile phase for equal gradient elution. Multiple reaction monitoring (MRM) in positive ion mode was used, and qualitative confirmation was achieved from retention time and secondary mass characteristic ions of TTX. The matrix-matched external standard calibration curves were used for quantitative analysis. Results Tetrodotoxin and impurity could be well separated, and it showed good linearity in the range of 0.10-2.0 mg/kg (r=0.997). The detection limit of the method was 0.012 mg/kg, the limit of quantification was 0.041 mg/kg, the average recovery was 75.7%-108.1%, and the precision was 1.4%-5.1%. The method was applied to 20 fishes, while TTX was not detected.Conclusion The method was accurate, efficient and simple, and could be applied to the recognition and identification of factors of food poisoning.