The invention provides a separating and culturing method for olfactory ensheathing cells of rats. The separating and culturing method comprises the following steps: (1) disinfecting a tissue processing instrument; (2) selecting male SD rats of 2-3 months old, killing the male SD rats after anesthesia, separating the olfactory mucosa tissue by adopting surgical scissors, placing the tissue in PBSsolution which is precooled, is sterile and contains double antibodies, and washing the tissue for a plurality of times, so that blood stains and outer membranes are removed; (3) cutting up the tissue, and carrying out digestion by adopting preheated neutral enzyme and trypsin respectively; (4) terminating the digestion, carrying out centrifugation, and discarding the supernatant; (5) inoculatingthe obtained product into an enveloped culture flask by adopting an artificial wall sticking method; (6) carrying out incubation for 1-2 h, and carrying out culturing in the environment being at 37 DEG C containing 5% CO2; (7) carrying out cell purification; (8) carrying out cell morphological observation; (9) carrying out cell subculturing; (10) carrying out cell counting and survival rate testing; and (11) carrying out immunological identification. For the separating and culturing method for olfactory ensheathing cells of rats, the operation is simple, a large number of cells are obtained, the survival rate is high, the method belong to an ideal primary separating and culturing method for the olfactory ensheathing cells of rats, and a reliable cell resource is provided for the experiment.