Abstract: This experiment was to optimize the extraction process of water chestnut shell flavonoids and investigate its inhibitory effect on the proliferation of human cervical cancer Hela cells. A single factor experiment was used to investigate the effects of extraction time, material-to-liquid ratio, ethanol concentration and extraction temperature on the yield of total flavonoids in water chestnut shells, and response surface analysis was carried out based on the yield of total flavonoids in water chestnut shells to obtain the extraction of water chestnut shell flavonoids. At the same time, the CCK-8 method was used to determine the effect of water chestnut flavonoids on the proliferation of human cervical cancer Hela cells, and to determine its IC₅₀ value to investigate the anti-tumor activity of water chestnut flavonoids. The experimental results showed that the best extraction process of water chestnut shell flavonoids was: extraction time 28 min, material-to-liquid ratio 1:33 g/mL, and ethanol concentration 53%. Under these conditions, the extraction rate of total flavonoids in the crude extract of water chestnut shell could reach 3.455%±0.16%. Incubation with the crude extract or purified solution of water chestnut shell flavonoids at concentrations of 5, 100, 200, 400, 800 µg/mL, all had a proliferation inhibitory effect on Hela cells, and there was a time and concentration dependence. Taking action time 48 h as an example, the IC₅₀ values ​​of the crude extract of water chestnut shell flavonoids, the purified material of water chestnut shell flavonoids, and 5-Fu were 271.46, 268.16, and 152.09 µg/mL, respectively. Therefore, at the same time, the inhibitory effect of water chestnut extract on the proliferation of Hela cells was weaker than that of the positive control 5-Fu.