The uncertainty of determination of four organophosphorus pesticide residues in fish by high performance liquid chromatography-tandem mass spectrometry was evaluated. According to GB 31656.8-2021 National Food Safety Standard-Determination of organophosphorus pesticide residues in aquatic product-Liquid ChromatographyTandem Mass Spectrometry, the residues of fenthion, malathion, dichlorvos and coumaphos in fish were determined,and the measurement uncertainty was evaluated by the data confirmed by the method. Establish mathematical model, analyze and quantify factors such as sample weighing, purity of standard, measuring instruments, standard curve fitting, recovery rate, precision, instruments and equipment, etc. When the contents of fenthion, malathion, dichlorvos and coumaphos in the sample were 46.43, 94.96, 40.91, 94.34 μg/kg, the expanded uncertainties were 8.90, 18.49, 8.27, 20.16 μg/kg, respectively. The evaluation results show that the uncertainty of this method mainly comes from standard curve fitting, measuring instruments, precision and recovery rate, and the evaluation results can provide a basis for scientific evaluation of the accuracy of the measurement results of organophosphorus drug residues in fish.