Chick embryo extract (CEE) contains a variety of growth factors which may improve in vitro follicle growth. Therefore, the effect of CEE on mouse pre-antral follicle culture was evaluated. Different percentages of CEE (0, 0.50%, 1.00%, 5.00% and 10.00%) were added to culture medium. Hence, the osmolarity of media was measured. Pre-antral follicles with diameter of 120-150 μm were isolated from 12-14 days old mouse ovary and cultured for 12 days. After culture, the maturation rate was assessed. Granulosa cells viability was evaluated using MTT test and estradiol levels were evaluated using related radio-immunoassay (RIA). Genes expression (BMP15 and ALK6) was also evaluated. The osmolarity of media and granulosa cells viability were the same in all groups. Estradiol level in group with 10.00% CEE was significantly decreased compared to the control group. After 12 days culture, the percentage of antral follicles development was significantly higher in the group with 5.00% CEE compared to control group. The percentage of metaphase II and germinal vesicle breakdown oocytes was significantly higher in group 5.00% CEE compared to control group. The expression of BMP15 gene in antral follicles in 5.00% CEE and control groups was significantly lower compared to pre-antral follicles. However, the expression of ALK6 gene in antral follicles in 5.00% CEE and control groups was not significantly different compared to pre-antral follicles. The increasing effect of CEE on follicle viability with keeping normal gene expression indicates that addition of proper percentage of CEE to culture media improves culture conditions, making it a possible choice to be used as a follicular growth enhancer in infertility clinics.