Aim: The aim of this study is to establish a method for determination of eupatilin in Folium artemisiae Argyi and observe the inhibitory effect of Folium artemisiae Argyi extract on human hepatoma SMMC-7721 cells. Methods: High-performance liquid chromatograph system with 2910 pump, 2930 UV detector, and N2000 workstation was used for determination of eupatilin in Folium artemisiae Argyi. Human hepatoma SMMC-7721 cells were cultured and cell proliferation was measured using the MTT assay. The expression protein levels of p53, Topo II, and bcl-2 were detected using Western blotting. Results: Eupatilin exhibited a linearity range of 0.5–3.0 μg/mL and a recovery of 100.72%, relevant standard derivation = 2.28%. Folium artemisiae Argyi extract had marked cytostatic and cytotoxic effects on SMMC-7721 cells, inhibited the SMMC-7721 colony formation in a dose-dependent manner. Folium artemisiae Argyi extract already possessed delayed effect after treating SMMC-7721 cells for 8 h, which became obvious at 12 h from treatment. After drug withdrawal, cells still tended to apoptosis. Folium artemisiae Argyi extract could inhibit p53, Topo II, and bcl-2 expressions in tumor cells. The present method for determination of eupatilin is simple, fast, accurate, sensitive, and reproducible. Conclusion: Hepatoma SMMC-7721 cells are quite sensitive to Folium artemisiae Argyi extract, which may be associated with its suppression of p53, Topo II, and bcl-2 expressions.