The invention discloses separation equipment for cell exosome and a using method of the separation equipment, and belongs to the technical field of cell separation. The separation equipment comprises a shell, wherein an end cover is hinged to the top of the shell, a rotating disc is arranged at the middle of the interior of the shell, and inserting grooves used for containing a test tube containing cells are formed in the rotating disc; and a lifting mechanism for controlling the rotating disc to lift is arranged below the rotating disc. By arranging the lifting mechanism, a driving mechanism, a linkage mechanism, a sealing mechanism and a limiting mechanism, when an experimenter opens the end cover, the linkage mechanism and the lifting mechanism are used for lifting the rotating disc, the experimenter can conveniently take and place the test tube, when the end cover is closed, the rotating disc descends to a working position, and then the upper end of the rotating disc is sealed through the sealing mechanism; and finally, the end cover is limited through the limiting mechanism, the experimenter does not need to take out the rotating disc and seal the test tube after the test tube is placed, the operation of the experimenter is facilitated, and the preparation time before an experiment is saved.