To establish a method for determination of mulberroside A content in Ramulus Mori; and to study the antitumor effect and mechanism of Ramulus Mori extract. HypersiI ODS column (4.6 mm × 250 mm, 5 μm) was used. Mobile phase was methanol-water (22:78), detection wavelength was 303 nm, column temperature was 25, and flow rate 0.8 mL/min. Mouse model of HepA tumor was established to observe the effect of Ramulus Mori extract on tumor growth. Serum IL-2 and TNF-α level in mice were detected by radioimmunoassay. Mulberroside A presented a good linearity within 0.064-0.640 μg (r=0.9998); average recovery was 101.2%; and mulberroside A content in Ramulus Mori extract was 5.16%. Ramulus Mori extract inhibited the growth of HepA tumor in mice and promoted the production of IL-2 and TNF-α. The method proposed is accurate, simple and fast, which can be used for determination of mulberroside A content in Ramulus Mori extract. Ramulus Mori extract may exert antitumor effect by promoting the production of IL-2 and TNF-α.