Objective：In mouse embryonic stem cells（mESC），proximity labeling technology based on engineered ascorbate peroxidase（APEX2）and mass spectrometry were used to identify nucleolar related proteins. Methods：An APEX2-FBL overexpression stable cell line was constructed，biotin-labeled protein was obtained by APEX2 proximity labeling technology，the biotin-labeled protein was enriche with magnetic beads coated with streptavidin，and then liquid chromatography-tandem mass spectrometry（LC-MS/MS），and bioinformatics analysis was used to screen differential proteins. Results：Highly expressed APEX2-FBL stable cell lines were suuessfully constructed；Western blot results showed that a large number of biotin-labeled proteins were obtained by APEX2 proximity labeling technology；837 differential proteins were identified by mass spectrometry，and the biological processes were mostly related to nucleoli. Conclusion：The use of adjacent biotin labeling technology combined with mass spectrometry is a feasible method to identify nucleolar related proteins.