The invention discloses a separation culture method of laying duck small yellow follicle granular cells. The separation culture method includes following steps: selecting a laying duck, killing the laying duck by bloodletting a jugular vein, respectively using bromogeramine diluent and alcohol to disinfect the body of the laying duck, and separating follicles of 3-8 mm in an ovary; stripping outer-layer blood vessels and connective tissue of the follicles, fixing the follicles, discharging yolk, stripping granular cell layers, and cleaning before centrifuging to abandon supernate, adding an enzymolysis solution for digestion, sieving, cleaning with an M199 culture medium, centrifuging to abandon supernate; adding red blood cell lysis liquid, and centrifuging to abandon supernate; adding an M199 culture medium containing fetal calf serum, suspending granular cells, adjusting density of suspension cells, and inoculating for culture to obtain the laying duck small yellow follicle granular cells. The separation culture method is suitable for both separation of granular cells in big yellow follicles of ducks and separation of small yellow follicle granule cells; the granular cells are high in purity and activity and stable in cell function; the separation culture method has the advantages of simple operation, easiness in repeating and stable result.