Study on the stem chemical composition and in vitro anti-tumor activity of three perennial medicinal herbaceous plants, namely Begonia palmata, Rubia ovatifolia and Boehmeria nivea. Extract and separate ethanol extracts from three plants, systematically and deeply identify and analyze the physicochemical properties of plant ethanol extracts using GC-MS and UPLC-QTOF-MS methods, and evaluate the in vitro anti-tumor activity of compounds using MTT colorimetric method. GC-MS detected 28 compounds in Begonia palmata, 23 compounds in Rubia ovatifolia, and 24 compounds in Boehmeria nivea. The killing rates of three plant summer stem extracts on NCL-H1703 lung squamous cell carcinoma cells were ranked from high to low as Begonia palmat (52.77%)>Rubia ovatifolia (51.26%)>Boehmeria nivea (50.55%). The findings of this study not only enhance the chemical database of three plant stem metabolites, but also confirm their anti-tumor material basis, and provide candidate compounds and network pharmacological target reference for the development of new plant-derived anti-tumor drugs.