Prunus mume is an ancient medicinal herb and food that are commonly used in Asian countries with high nutritional ingredients and biological activities. Polyphenols are important functional components in Prunus mume. To obtain a more efficient extraction process of Prunus mume polyphenols, a single-factor test and response surface method were used. After extraction and purification, the final polyphenol content of Prunus mume (L1) was up to 90%. Biological experiments showed that L1 had high anticancer activity against HeLa (125.28 μg mL-1), HepG2 (117.24 μg mL-1), MCF-7 (170.19 μg mL-1), and A549 (121.78 μg mL-1) in vitro by MTT assay. The combination of DDP and DOX significantly enhanced the anticancer activity of the four cell lines, especially L1-DOX had the smallest IC50 value of 0.04 μg mL-1 against HepG2 cells, indicating the combination of drugs had synergistic effects. It is further demonstrated that L1 could inhibit cell proliferation by inducing apoptosis with ROS detection and confocal fluorescence images. The relative tumor proliferation rate (T/C) was 40.6%, and the tumor inhibition rate was 57.9%, indicating L1 to have no significant toxicity but high anti-HepG2 activity in vivo. Although the study is very limited, it is anticipated to provide a reference for further exploration of the functionality of the plant.