Objective To explore a suitable separation protocol for exosomes derived from urine of patients with aortic dissection. Methods Urine samples were collected from the patients with aortic dissection. The exosomes were extracted and purified by ultracentrifugation, dialysis-ultrafiltration and precipitation kit methods. The biological morphology of exosomes was observed under transmission electron microscope. The particle size, distribution and concentration of the exosomes were detected by nanoparticle tracking analysis. Results The exosomes extracted with ultracentrifugation method had half cup-like or concave-hemispherical morphology; the particle size was large and well-distributed; the range of the particle size was wide with a mean value of (236.4±46.5) nm; and the concentration was low, being (2.82±0.21)×10¹² per 1 mL urine. The exosomes extracted by dialysis-ultrafiltration method had normal morphology and large quantity; the particle size was small and concentrated; the range of the particle size was narrow with a mean value of (128.7±6.3) nm; and the concentration was high, being (2.16±0.15)×10¹⁴ per 1 mL urine. There were no exosomes in the extractive by precipitation kit method under transmission electron microscope. Conclusion Dialysis-ultrafiltration method is a suitable method for extracting exosomes from urine of the aortic dissection patients and can yield a high concentration, while it is not suitable when exosomes of very high purity are required.