Inspired by the common structural characteristics of numerous known antitumor compounds targeting DNA or topoisomerase I, 3-(benzazol-2-yl)-quinoxaline-based scaffold was designed via the combination of two important privileged structure units —quinoxaline and benzazole. Thirty novel 3-(benzazol-2-yl)-quinoxaline derivatives were synthesized and evaluated for their biological activities. The MTT assay indicated that most compounds possessed moderate to potent antiproliferation effects against MGC-803, HepG2, A549, HeLa, T-24 and WI-38 cell lines. 3-(Benzoxazol- -2-yl)-2-(N-3-dimethylaminopropyl)aminoquinoxaline (12a) exhibited the most potent cytotoxicity, with IC₅₀ values ranging from 1.49 to 10.99 μM against the five tested cancer and one normal cell line. Agarose-gel electrophoresis assays suggested that 12a did not interact with intact DNA, but rather it strongly inhibited topoisomerase I (Topo I) via Topo I-mediated DNA unwinding to exert its anticancer activity. The molecular modeling study indicated that 12a adopt a unique mode to interact with DNA and Topo I. Detailed biological study of 12a in MGC-803 cells revealed that 12a could arrest the cell cycle in G2 phase, inducing the generation of reactive oxygen species (ROS), the fluctuation of intracellular Ca²⁺, and the loss of mitochondrial membrane potential (ΔΨm). Western Blot analysis indicated that 12a-treatment could significantly up-regulate the levels of pro-apoptosis proteins Bak, Bax, and Bim, down-regulate anti-apoptosis proteins Bcl-2 and Bcl-xl, and increase levels of cyclin B1 and CDKs inhibitor p21, cytochrome c, caspase-3, caspase-9 and their activated form in MGC-803 cells in a dose-dependent manner to induce cell apoptosis via a caspase-dependent intrinsic mitochondria-mediated pathway. Studies in MGC-803 xenograft tumors models demonstrated that 12a could significantly reduce tumor growth in vivo at doses as low as 6 mg/kg with low toxicity. Its convenient preparation and potent anticancer efficacy in vivo makes the 3-(benzazol-2-yl)quinoxaline scaffold a promising new chemistry entity for the development of novel chemotherapeutic agents.