A new series of bis-benzimidazole derivatives was designed and synthesized. In vitro cytotoxity evaluation showed that these compounds exhibited high activity against the selected tumor cells. Among them, compound 8 owned the best potential, its IC50 values being 0.58 μM (mononuclear tumor cell line (U937)) and 0.56 μM (cervical cancer cell (Hela)), respectively. Molecular modeling, fluorescence and viscometry study showed that compound 8could bind into the minor groove of DNA.