Chronic myelogenous leukemia (CML) is an uncommon type of cancer of the bone marrow associated with high mortality. Although several effective therapies have been developed to reduce symptoms in patients with CML, many of these methods are associated with side effects. Coreopsis tinctoria Nutt. (C. tinctoria) is a natural medicinal material that possesses antioxidant and anticancer activities. Yet, its effect in treating leukemia has still not been fully explored. To optimize the C. tinctoria flower extraction process and investigate whether these extracts can impair CML cell survival. The extraction process of C. tinctoria was optimized by the Box-Behnken design response surface method. K562 cells were treated with different volumes (0, 10, 25, 50, and 100 μL) of C. tinctoria flower extracts. The effect of C. tinctoria extract on cell morphology and cell apoptosis was assessed by light microscopy, laser confocal microscopy, and flow cytometry. We established the following optimized C. tinctoria flower extraction conditions: temperature of 84.4°C, extraction period of 10 mins, solid-liquid ratio of 1:65, and times 4. These conditions were applied for C. tinctoria flower extraction. Pre-incubation of extracts prepared under the aforementioned optimal conditions with K562 cells induced cell cytotoxicity and cell apoptosis. C. tinctoria flower extracts exert obvious anti-leukemia effects in vitro and may be a potential drug candidate for leukemia treatment.