CD63 is a ubiquitously expressed transmembrane glycoprotein frequently used as a general protein marker of extracellular vesicles (EVs). EVs are cell-secreted and membrane-enclosed nanoparticles which circulate through the blood and other body fluids and thus are available for isolation. EV cargo is very dynamic and reflects the biological status of the originating tissue, which, coupled with their accessibility from body fluids, could make EVs a valuable diagnostic and prognostic tool. However, identification of tissue-specific EVs is still lacking. The goal of this study was to examine the tissue specificity of CD63 protein using a novel murine monoclonal antibody mCD63.07 produced at the Center for Proteomics, University of Rijeka, Faculty of Medicine, Croatia. Western blot analysis of salivary gland, brain, lung, liver, kidney, muscle, bone marrow and spleen tissue obtained from wild type (wt) mice revealed that the expression levels and protein forms of CD63 varied among murine tissues. Molecular weight of detected CD63 forms ranged from 25 to 170 kDa, depending on the tissue, whereby some of CD63 forms were tissue-specific. A comparison with corresponding knockout tissues confirmed the high specificity of mCD63.07 antibody. PNG-ase F deglycosylation treatment of proteins from salivary gland, kidney, bone marrow, liver, muscle and spleen wt samples resulted in merging of higher molecular weight protein bands into one band at approximately 24 kDa. Thus the observed differences in detected CD63 forms arise from tissue-specific N-glycosylation of the CD63 protein. The findings indicate that CD63 glycoforms are tissue-specific and therefore might be used for isolation of tissue-specific EVs. Future characterization of human tissue-specific glycoforms of CD63 could enable the development of new EV-related blood-based diagnostic methods.