ObjectiveThe study aimed to evaluate the measurement uncertainty in the determination of five cacaine anesthetics and their metabolites residues by high-performance liquid chromatography-tandem mass spectrometry （LC-MS/MS）. MethodsThrough the establishment of mathematical model， the sources of uncertainty were analyzed， and various components were quantified and synthesized to evaluate the influence of the uncertainty components on the measurement results． ResultsThe uncertainties of the experiments were mainly derived from calibration curve fitting， sample pretreatment， recovery rate， standard solution preparation and measurement repeatability. Furthermore， the expanded uncertainty related to the content in shrimp （k=2） was 1.18 μg‧kg^-1 at 8.68 μg‧kg^-1 for tricaine methanesulfonate （MS-222）， 1.28 μg‧kg^-1 at 9.11 μg‧kg^-1 for benzocaine， 13.5 μg‧kg^-1 at 91.4 μg‧kg^-1 for 4-aminobenzoic acid， 12.2 μg‧kg^-1 at 91.0 μg‧kg^-1 for p-acetylamino benzoic acid， and 11.3 μg‧kg^-1 at 95.3 μg‧kg^-1 for 3-aminobenzoic acid， respectively. ConclusionThis method is suitable for the uncertainty analysis of cacaine anesthetics and their metabolites determination in aquatic products by LC-MS/MS， and can provide scientific and reliable basis for the measurement accuracy.