Firstly, the antioxidant capacity of total flavonoids that was extracted from Sorbus pohuashanensis (Hance) Hedl was determined by ABTS+·radical-scavenging method, and MTT assay was used to mensurate the anti-tumor activity of total flavonoids In Vitro. Subsequently, the extraction conditions of flavonoids were optimized by the response surface methodology, the antioxidant activity was investigated simultaneously. Furthermore, the anti-tumor activity mechanism of flavonoids was simulated by molecular docking software. The antioxidant activity of flavonoids was correlated with the content in the process of optimizing the extraction condition. Moreover, The MTT assay revealed that SPF inhibited the activity of four sorts of cancer cells and positively correlated with the concentration of total flavonoids. And the experimental data demonstrated its anticancer activity was better at high concentrations. Finally, its anti-tumor activity was found that may be related to small molecules from flavonoids bind the apoptotic proteins Caspase-3, Caspase-8, and Caspase-9. The study could provide a theoretical basis for further pharmacodynamic research of Sorbus pohuashanensis (Hance) Hedl flavonoids, and improved a valuable attribute to Sorbus pohuashanensis (Hance) Hedl.