The invention discloses a method for analyzing the purity of Fmoc-protected amino acids and related substances, comprising the following steps: S1, preparing a Fmoc-Osu impurity control solution, so that the ratio of Fmoc-Osu to Fmoc-protected amino acids is 0.1-1.0:100, and obtaining Fmoc-Osu impurity contrast solution; S2, prepare relevant impurity contrast solution, make the ratio of single relevant impurity and Fmoc-protected amino acid be 0.1-1.0:100, obtain steps such as multiple reference substance solutions, the present invention passes mobile phase, impurity The control solution and gradient elution conditions were comprehensively designed and optimized. During the operation of the system, the analysis method of the present invention has a stable baseline, basically no interference peaks in the blank test, outstanding impurity separation effect, and good specificity. The test data proves that the detection The limit can reach 0.002%, and the sensitivity is extremely high. The test results can provide reliable and effective information for product development, production and purification, lay a solid foundation for the establishment of quality control standards for the protection of amino acids, and play a positive role in the quality control of the final product.