To establish a method for the determination of morpholine guanidine residues in lettuce, ultra performance liquid chromatography-tandem mass spectrometry and uncertainty evaluation of the established method were studied. After samples were extracted and purified, the morpholine guanidine was quantified by scanning with positive ions from an electrospray ionization source in multiple reaction monitoring mode. The uncertainty was synthesized by building a mathematical model to analyze the sources of measurement uncertainty and quantify each uncertainty component. The morpholinoguanidine showed a good line in the range of 0.1~20 mg/L with a correlation coefficient R2 of 0.999 3. The spiked recoveries at the three levels of 0.5, 5.0 and 10.0 μg/kg ranged from 84.08% to 98.06% (n=6) with the relative standard deviations of 3.58% to 5.26% (n=6), and the extended uncertainty was 2.84 μg/kg (k=2) at 95% confidence interval for the determination of morpholine guanidine in the sample at 17.5 μg/kg. The method was rapid and efficient, highly accurate and reproducible, and the recovery and precision can meet the test requirements. The uncertainty was mainly caused by the standard solution, the establishment of the standard curve and the recovery of the sample spiking. The experimental results could provide a reference for the determination of morpholine guanidine residues in lettuce by UPLC-MS/MS and the uncertainty evaluation.