The invention discloses a separation, primary culture and subculture method for granulosa cells in follicles in a pig ovary GV period. The method comprises the following steps: collecting a cyst-free healthy pig ovary, and cleaning the ovary; separating granular cells; carrying out primary culture on the granular cells for 48 hours; changing liquid in a culture bottle for primary culture; after changing the liquid, continuously culturing the cells for 36 hours, and cleaning, digesting and terminating the cultured cells; and finally, sub-packaging and subculturing the cells for 48 hours. The invention provides a pollution-free ovary collection method, optimizes a granulosa cell acquisition and culture method, and further provides a primary granulosa cell subculture method. Granular cells obtained by the method are almost free of pollution, high in cell uniformity, high in consistency in the growth and development period, high in cell survival rate, high in success rate of making oxidative stress models and the like, and high in result consistency, practicability, stability and feasibility. In addition, the number of obtained cells is large, many subsequent experiments can be carried out, the number of times of going to a slaughter house is reduced, and the efficiency is high.