Objective In order to analyze of poisoning causes, a new method was established utilizing hydrophilic liquid chromatography-tandem triple quadrupole mass spectrometer (HILIC-MS/MS) coupled with dispersive solid phase extraction for rapid qualitative and quantitative analysis of tetrodotoxin in nassarius and shellfish. Methods Sample(1.0 g)was extracted with 0.1% acetic acid in boiling water bath, purified by dispersive solid phase extraction with 50 mg hydrophilic-lipophilic balance (HLB), 5 mg graphitized carbon black (GCB) and protein precipitation with acetonitrile, and then filtered through a polytetrafluoroethylene(PTFE)membrane. The analytes were separated on a HILIC column, and detected in selected reaction monitoring (SRM) mode via positive electrospray ionization. The matrix matching and external standard method was used for quantification.Results Tetrodotoxin showed good linearity in the concentration range between 2.0 and 40.0 ng/ml, the correlation coefficient was higher than 0.999. The detection limit of tetrodotoxin in seafood was 10.0 μg/kg. The rates of recovery varied between 74.2% and 87.9% with relative standard deviations from 2.3% to 9.1% at spiked concentrations of 25,0 and 200 μg/kg. The proposed method was applied in the detection of tetrodotoxin in shellfish and nassarius from coastal cities of Zhejiang Province. Conclusion The method was accurate, fast, easy to operate, which could meet the requirements of public health emergency testing or routine testing.