The extracellular matrix (ECM) is the connective tissue component generated and secreted by cells to provide structural and functional support, while extracellular vesicles are distinct membrane-enclosed structures present outside of eucaryotic cells that carry out distinct biological functions. Different cell types release distinct populations of vesicles that fulfill various functions. Exosomes are vesicles commonly secreted by a variety of cells, whereas matrix vesicles (MVs) are specifically produced and secreted by bone cells to facilitate the formation of the ECM. This article focuses on the characteristics of the ECM and extracellular vesicles, and reviews the latest progress in applying proteomic technologies to analyze these features. The findings and implications in developmental biology, tumor biology, immunology, biomarker discovery, and vaccine research are also discussed.