Milk-derived exosomes contains protein and RNA, which mediates the communication between cells information exchange and materials transfer. Objectives were to isolate and characterize exosomes in Jersey cattle milk. Milk samples were collected from 12 mid-lactation Jersey cows and 12 Holstein cows as comparison. We separately use 30 mL milk at a time to isolate. Milk fat was removed by centrifugation twice at 1,200 × g, 4°C,10 min. Milk protein and cell debris were also removed by centrifugation at 21,500 × g, 4°C for 30 min and 1 h, respectively. Supernatant (whey) was sequentially filtered through 0.45-µm and 0.22-μm filters. The whey samples were ultracentrifuged twice at 100,000 × g, 4°C for 90 min to isolate exosome. Exosomes obtained in the pelleted were resuspended in 100 µL PBS. Exosomes samples were diluted in PBS. Ten-microliter exosome samples were applied to 100 mesh copper grids and precipitated for 2 min, respectively. Excess material was removed by blotting and samples were negatively stained twice with 10 μL of a 2% uranyl acetate solution (wt/vol; Electron Microscopy Services). The grids were air-dried and exosomes were visualized using a transmission electron microscope at 100 kV (Hitachi H-7500, Hitachi, Japan). The particle size distribution and zeta potential were determined with a Zeta VIEW S/N 17–310 instrument (Particle metrix, Germany) at 4°C. The exosomes were diluted 6,000 times with ultrapure water to do nanoparticle tracking analysis. Exosomes marker proteins were detected by in-gel enzymatic hydrolysis. Transmission electron microscope images of exosomes isolated from Jersey cattle milk had a diameter in the range of 30–150 nm. Peak intensities for isolated exosomes were observed at 114.6 nm from nanoparticle tracking analysis. Two high abundance proteins, HSP70 and CD63, were identified in Jersey cattle milk-derived exosomes by In-gel enzymatic hydrolysis. These preliminary results indicate that exosomes from Jersey cattle milk has similar morphology to exosomes isolated with Holstein cow. The functional properties (e.g., miRNA, protein) in isolated exosomes merits further investigations.