【Objective】To explore the impact of Chinese herbal formula “Huirukang” on serum estrogen(E), progesterone(P4) and transforming growth factor β1(TGF-β1) levels in dairy cows during the milk withdrawal period.【Method】Eighty healthy cows in late pregnancy with (15.42±0.71) kg daily milk yield were randomly divided into four groups: control group and Chinese medicine groups（fed 400, 500 and 600 g “Huirukang” daily）. The method of gradual milk cessation was used. We collected venous blood from the cow tail on day 0, 1, 3, 5, 7, 9 and 11, and recorded the daily milk yield. ELISA was used to determine the expression levels of serum E, P4 and TGF-β1.【Result】Control group and Chinese medicine groups (400, 500, 600 g) dried off on day 11, 7, 5 and 5 respectively. Serum E levels declined in all four groups, and the decline in control group was slower compared to Chinese medicine groups. P4 level did not change significantly during the milk withdrawal period for control group. P4 levels increased first in Chinese medicine groups, then declined after the milk withdrawal period, and by the end of the test period were still significantly higher compared to control group. TGF-β1 levels increased in all four groups during the milk withdrawal period, and declined after the milk withdrawal period in Chinese medicine groups. The levels of E, TGF-β1 and daily milk yield showed significant correlation between each other during the milk withdrawal period, while P4 had no significant correlation with E, TGF-β1 or daily milk yield.【Conclusion】During the milk withdrawal period, Chinese herbal formula “Huirukang” can significantly promote cows to dry off. E is a negative regulation factor for milk withdrawal, while TGF-β1 is a positive regulation factor. P4 has no significant effect on milk withdrawal.