Extracellular vesicles (EVs) are released by virtually all cells, carry cellular molecules to the extracellular environment, and may interact with other cells. They are found in body fluids, therefore, constituting useful target sources for the identification of disease biomarkers, for example, in cancer. EVs originate from the plasma membrane or from multivesicular endosomes. They have the same topology as the plasma membrane and are rich in glycoconjugates, displaying specific glycosignatures. Surface glycoconjugates play important roles in EVs biogenesis and in their interaction with other cells. Changes in glycosylation constitute a hallmark of different types of cancer, therefore, the study of glycoconjugates and glycosignatures of EVs appear as promising candidates to identify novel cancer biomarkers and to increase the specificity and sensitivity of the existing clinical biomarkers, many of which are glycosylated.