The aim of the present study was to optimize flavonoid extraction from Chrysanthemum morifolium and to study the antitumor effects of flavonoids on human gastric cancer MKN45 cells in vitro. A single factor experiment was designed and the extraction process was optimized using an orthogonal test. MKN45 cells were treated with different concentrations of flavonoid from Chrysanthemum morifolium for 24 and 48 h and the inhibitory effect on the MKN45 cells was evaluated using an MTT assay. Following staining with Annexin V-fluorescein isothiocyanate/propidium iodide, flow cytometry was performed. The optimized flavonoid extraction conditions were as follows: Duration of ultrasonic treatment: 35 min; ethanol concentration: 75%; extraction temperature: 80°Cand liquid-to-solid ratio 25: 1. Under the above conditions, the extraction rate of flavonoids was 5.24%. When compared with a blank control group, flavonoids extracted from Chrysanthemum morifolium inhibited the proliferation of MKN45 cells in a dose- and time-dependent manner. Furthermore, in cell groups treated with low, moderate and high concentrations of flavonoid, it was observed that the proportion of apoptotic cells increased in a dose-dependent manner. The extraction process optimized by the orthogonal test achieved a high yield and satisfactory extraction efficiency. Additionally, the experiment demonstrated that flavonoids from Chrysanthemum morifolium inhibited the growth of MKN45 cells and induced their apoptosis. Thus, flavonoids from Chrysanthemum morifolium exerted antitumor effects on MKN45 cells, which may be exploited as a potential antitumor therapeutic for gastric cancer.