Stephania cepharantha Hayata is a traditional Chinese herbal medicine used to treat lung cancer, and its alkaloids, especially cepharanthine (CEP), were reported to be its effective ingredients. Therefore, the extraction of potential antitumor ingredients from the plant was of interest. We first explored the optimized solvent extraction of antitumor agents from S. cepharantha Hayata guided by an in vitro antitumor activity assay. The solvent for extraction and its concentration, the liquid to material ratio, extraction duration, particle size, macerating time, and the frequency of extraction were investigated using a single-factor experiment. An orthogonal design (L9, 3⁴) was constructed to determine the suitable extraction conditions. The crude extract was then purified sequentially by macroporous adsorption resins (MR) for the enrichment of CEP. Under these optimal conditions, the yield of total alkaloids in the herbs was 3.4%, whereas the CEP content was 2.9%. Total alkaloids exhibited significant anti-proliferative activities in the A549 cell line. Our study provides means for the further development and use of the antitumor components from S. cepharantha, which has potential for application in the pharmaceutical industry.