This study was aimed at identifying a cytotoxic compound on human acute lymphoma jurkat T cells from bark of Albizzia julibrissin Durazz and determining its synergistic effect with doxorubicin. The partially purified substance from A. julibrissin, HaBC18, was further purified through recycling HPLC chromatography. The purified cytotoxic substance of A. julibrissin was estimated to have about 94.5% purity by analytical HPLC and designated “Ha75F1”. Chemical structure of the Ha75F1 was analyzed with Mass spectrum, 1H-NMR, 13C-NMR, HMBC and HMQC spectrum. The Ha75F1 is identified as Julibroside III. Its molecular formula is 12C10313CH164NO48 and its molecular weight is 2,219Da. The mixture with Julibroside III and doxorubicin led to enhanced cytotoxic effect on jurkat T cell. In conclusion, the Julibroside III purification method from A. julibrissin was established and its strong cytotoxicity was evaluated. The mixture of Julibroside III and doxorubicin represented stronger anticancer activity than each compound on jurkat T cell. Thus, this material has great potential as an anti-tumor agent, health promotion or tonic agent.