This study aims to investigate the protein contents of serum-EVs associated with diffuse idiopathic skeletal hyperostosis (DISH) and identify the potential biomarkers for the diagnosis of DISH. Serum-EVs were separated using differential centrifugation technique. Label-free-liquid chromatography mass spectrometry (LC-MS/MS) was utilized to identify differentially expressed proteins (DEPs) highly associated with DISH. DEPs’ expression levels were tested using Enzyme-linked immunosorbent assay (ELISA) and bioinformatic analyses were performed.