Objective To evaluate the uncertainty during the determination of sulfur-containing amino acids in goat milk powder by an amino acid analyzer.Methods The mathematical model and fishbone diagram of the uncertainty source are established to analyze and quantify the introduced uncertainty components. Then, the combined uncertainty and extended uncertainty are calculated.Results When the mass fractions of cystine, cysteine, and methionine are 0.305, and 0.338 g/100 g, the extended uncertainty is 0.022, and 0.023 g/100 g, respectively (k=2).Conclusion The uncertainty of this method mainly comes from the weighing of standard material and samples, as well as the preparation of the standard series working solution, which should be strictly controlled during sample testing.