Objective To study the advantages of trypsin in isolating rat ovarian granulosa cells. Methods Ovarian granulosa cells of female SD rats aged 3-4 weeks were isolated and cultured by mechanical method and trypsin method, respectively. Mechanical method:under 40 folds anatomic microscope, mature follicles were punctured with a needle, and the culture solution containing the remaining tissue and follicular fluid of ovary was filtered with 200 mesh filter screen. Trypsin method:the ovary was cut into small pieces less than 2 mm³ with a sterile eye scissors, and then 0.25% trypsin was added. After digestion at room temperature for 1 h, the digestion solution containing the fragments of ovary was filtered with 200 mesh filter screen. The cell count, cell viability and secretion of estradiol and progesterone were compared. Results The survival rate of primary ovarian granulosa cells isolated by the two methods was both higher than 90%. When cultured for 9 d, the proliferation rate of the ovarian granulosa cells isolated by trypsin method was higher than that by mechanical method, except for 24 h. The peak time of cell proliferation rate of trypsin method was 24 h earlier than that of mechanical method, and the maximum cell proliferation rate of trypsin method was higher than that of mechanical method (72 h:[210.09±0.95]% vs 96 h:[180.50±0.74]%, P<0.05). The maximum secretion amount, total secretion amount and average daily secretion amount of estradiol and progesterone in ovarian granulosa cells isolated using trypsin method were significantly higher than that using mechanical method (all P<0.05). Conclusion Both cell viability and hormone secretion function of rat ovarian granulosa cells isolated by trypsin method are obviously superior to those by mechanical method. Trypsin method is worthy of further promotion in the study of premature ovarian failure.