Response surface methodology (RSM) was used to optimize extraction of flavonoids from Bombyx batryticatus. Variation of the flavonoids content over the course of 12 days, and flavonoids anticancer activities were evaluated. Significant extraction parameters were the ethanol concentration, the liquid-solid ratio, and the extraction temperature. The extraction time was not significant. Results of a polynomial regression model were in agreement with experimental results. Optimum conditions were an ethanol concentration of 80.89%, a liquid-solid ratio of 30.26, an extraction temperature of $100^{circ}C$, a time of 2.55 h, with a predicted TFC yield of 11.48mg of RE/g. Variation evaluation showed TFC changed in a certain rule with time. Flavonoids showed strong antioxidant activities, and suppressed proliferation of HeLa cells in a dose-dependent manner, but had no obvious effect on HEK293 cells, indicating a low toxicity.