The invention relates to a method for separating and preparing exosomes, belonging to the field of biotechnology. The method for separating and preparing exosome comprise that following steps: 1) removing cellular impurities by centrifugation; 2) centrifuging to remove cellular debris and large protein impurities; 3) transferring that supernatant into a new centrifugal tube, centrifuging to discard the precipitate and leave the supernatant; 4) filtering that supernatant and transferring; 5) mixing that reagent XBP and the sample, transferring the reagent XBP into a membrane adsorption column,centrifuging, and discarding the liquid at the bottom of the tube; 6) centrifuging to remove liquid remaining on that membrane; 7) adding reagent XWP, centrifuging, discarding that liquid at the bottom of the tube, and changing the column into a new centrifugal tube; and 8) adding that reagent XE to the column, centrifuging and eluting the exocrine body to obtain the liquid containing the exocrinebody. The RNA purity of the extracted exocrine body is improved, the impurity is less, the biological sample volume of the initially extracted exocrine body can be reduced, and the yield of the circulating exocrine body can be improved.