Exosomes are a specific subpopulation of extracellular vesicles that are widely released by cells of different origins with divergent functions that make their way into body fluids that can be conveniently sampled. In the current study, we isolated and evaluated exosomes from concurrently collected samples of milk, plasma, saliva, and urine from a group of 6 pregnant Holstein-Friesian dairy cows (aged 7 mo, 174 to 203 d of gestation). The cows had BCS of 3.5 to 5.25 (on a scale of 1 to 10), and the milk production for the season to the time of sampling ranged between 5,118 and 6,959 kg. The low levels of extracellular vesicles in saliva and urine (more than 86% fewer compared to the extracellular vesicles in milk and plasma) precluded further detailed evaluation since utility for diagnostics was deemed unlikely. In exosomes isolated from milk and plasma, size distribution, morphology, and the presence of exosome markers was confirmed by nanoparticle tracking analysis, electron microscopy, and Western blot. In addition, a targeted proteomic approach using the quadrupole ion trap mass spectrometer was also used in the study to screen for the exosome marker (e.g., Tumor susceptibility gene 101). Following confirmation of the presence of exosomes, the proteomic profiles of milk and plasma exosomes were evaluated using information-dependent acquisition-mediated liquid chromatography-tandem mass spectrometry (LC-MS/MS). The milk exosomes contain proteins that differed greatly from the plasma exosomes, with only 8 similar proteins harbored in both the milk and plasma exosomes. The milk and plasma exosomes were found to contain proteins (e.g., immunoglobulin J chain and α2 macroglobulin) associated with specific biological processes and molecular functions. Hence, the fluid of origin required for exosome analysis will be dependent on the specific information needed. In conclusion, isolated exosomes from milk and plasma samples collected at the same time point from the same dairy cows encapsulated different profiles of proteins associated with different biological processes and molecular functions.