Objective To establish a new screening system for protein-protein interaction using BioID combined with mass spectrometry, and to verify the interacting protein of Yes-associated protein 1 (YAP1) through co-immunoprecipitation and Western blotting. Methods The YAP1-BirA^* fusionexpression plasmid was constructed. After the plasmid was transfected into 293T cells, 50 μmol/L biotin was added to the cell culture medium, and the total proteins of the cells were extracted after 24 h of culture. The expression of YAP1-BirA^* fusionprotein was detected by Western blotting with YAP1 specific antibody. Horseradish peroxidase (HRP)-avidin was used to detect the level of proteins labeled by biotin in cells. The biotin-labeled proteins in cells were purified through affinity magnetic beads, and the proteins purified by affinity magnetic beads were dissolved after washing the nonspecific binding proteins on affinity magnetic beads for many times. A total of 40 μg total proteins were subjected to sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE), and then the proteins in the gel were stained by protein silver staining kit to determine the purification effect of affinity magnetic beads on biotinylated protein. Biotinylated proteins were identified by label-free mass spectrometry. For the identified proteins, the biotinylation of SMAD family member 3 (SMAD3) was verified by Western blotting. The interaction between SMAD3 and YAP1 was determined by co-immunoprecipitation and Western blotting. Results The YAP1-BirA^* fusionexpression plasmid was successfully constructed, which could efficiently expressed the fusion protein in 293T cells. In the presence of biotin, YAP1-BirA^* fusionprotein could biotin-label the proximity proteins close to YAP1-BirA^* fusionprotein in cells. After cell lysis, these biotin-labeled proteins in cells could be purified by magnetic beads coupled with avidin. After repeated washing, biotin-labeled proteins could be enriched. Mass spectrometry showed that the proteins close to YAP1 in space could be obtained. The interaction between SMAD3 and YAP1 was confirmed by co-immunoprecipitation and Western blotting. Conclusion BioID combined with mass spectrometry is a simple and efficient technology for protein-protein interaction screening. Combined with co-immunoprecipitation and Western blotting, it may become a new protein-protein interaction research system.