The content uncertainty of L-hydroxyproline in dairy products was evaluated by referring to SN/T 3929—2014 “Determination of L-hydroxyproline in export foods”. According to JJF 1059.1—2012 “Measurement uncertainty evaluation and expression”, and CNAS—GL006:2019 “The evaluation guidance for uncertainty in chemical analysis”, and these two existing standards for evaluation of uncertainty of the basic procedures, a mathematical model was established for the analysis of determination of L-hydroxyproline content in dairy products by high performance liquid chromatography tandem mass spectrometry, which introduced in the process of the measurement uncertainty components. The measured results of synthetic standard uncertainty and expanded uncertainty were obtained by calculation. The evaluation results showed that the uncertainty generated by fitting the standard work curve generated the greatest influence on the results, followed by the uncertainty caused by the measurement repeatability, and the uncertainty of sample weighing had the least influence on the results. The evaluation results can provide a reference for analyzing the accuracy of test results and the reliability of methods.