Abstract: In order to improve the conception rate of estrus synchronization-timed insemination technology in cows, this experiment studied the changes of reproductive hormone levels in the peripheral blood of Holstein cows after estrus synchronization and explored the optimal time for artificial insemination in Holstein cows. Twenty-four young barren Holstein cows were chosen for the experiment and treated with a combination of inhibin and estrus synchronization and timed insemination（TAI） program. Peripheral blood was collected before the first intramuscular injection of inhibin antigen and Buserelin（day 0）, the first intramuscular injection of PG（day 7）, the second injection of Buserelin（day 9）, and the first artificial insemination（day 10）. ELISA was used to detect the concentrations of AMH, inhibin（INH, represented by INHB）, follicle stimulating hormone（FSH）, and luteinizing hormone（LH） in peripheral blood of cows. The changes in reproductive hormone levels in peripheral blood of cows before and after estrus synchronization treatment, as well as the correlation and differences in reproductive hormone concentrations at different experimental stages, were analyzed. The results showed that the LH content at day 7 was extremly higher than at day 0, day 9 and day 10（P＜0.01）, among which, the LH content at day 9 was significantly higher than at day 10（P＜0.05） in the peripheral blood of cows. The FSH content at day 9 was extremly higher than at day 0 and day 10（P＜0.01）, the FSH content at day 7 was extremly higher than at day 10（P＜0.01） in the peripheral blood of cows. However, no significant change in AMH and INHB contents（P＞0.05） and the fluctuation of different hormone contents among different individuals was found, showing a relatively consistent level. The results of this experiment indicated that both INHB and AMH could play important roles in regulating the secretion of FSH during the follicular phase and promoting follicular development.