Cell-derived extracellular vesicles (EVs) are currently in the limelight as potential disease biomarkers. The promise of EV-based liquid biopsy resides in the identification of specific disease-associated EV signatures. Knowing the reference EV profile of a body fluid can facilitate the identification of such disease-associated EV-biomarkers. With this aim, we purified EVs from paired human milk and serum samples and used the MACSPlex bead-based flow-cytometry assay to capture EVs on bead-bound antibodies specific for a certain surface protein, followed by EV detection by the tetraspanins CD9, CD63, and CD81. Using this approach we identified body fluid-specific EV signatures, e.g. breast epithelial cell signatures in milk EVs and platelet signatures in serum EVs, as well as body fluid-specific markers associated to immune cells and stem cells. Interestingly, comparison of pan-tetraspanin detection (simultaneous CD9, CD63 and CD81 detection) and single tetraspanin detection (detection by CD9, CD63 or CD81) also unveiled body fluid-specific tetraspanin distributions on EVs. Moreover, certain EV surface proteins were associated with a specific tetraspanin distribution, which could be indicative of the biogenesis route of this EV subset. Altogether, the identified body fluid-specific EV profiles can contribute to study EV profile deviations in these fluids during disease processes.