Ovarian cancer is one of the leading causes of death in women. It is also an important factor for an increased worldwide gynecological death observed recently. Here, we have elucidated the effect and mechanisms of Gancao Nurish-Yin Decoction (GNYD) on the viability of ovarian cancer cells. Network pharmacology analysis was performed to explore the putative therapeutic targets of GNYD on the ovarian cancer cells. Experiments at the cellular and molecular levels using ovarian cancer cells were performed to further verify the effect of GNYD. According to the network pharmacology analysis, 282 genes related to the targets of GNYD were identified. Among them, 123 genes were found to be overlapping as the potential targets for treating ovarian cancer. Subsequent experiments proved that GNYD medicated serum has significantly inhibited the survival, growth and migration of the ovarian cancer cells. The number of apoptotic cells has increased after the treatment with GNYD medicated serum, and a higher proportion of cells were arrested at G2/M phase. Importantly, genes present in the AMPK-p53/p21 pathway were found to be significantly promoted. Thus, this study has not only demonstrated the potential therapeutic value of GNYD in inhibiting the survival, growth and migration of ovarian cancer cells in vitro, but also provide clues for further exploring the pharmacological and molecular mechanisms of Chinese medicine preparations acting on various types of cancer. © 2022 The Authors.