The enhancement of DNA affinity of small molecules usually ensures their high nuclease activities, and may also open a new scope of their applications in biology and medicine. In this work, we demonstrate that the nuclease activity and cytotoxicity of the small DNA intercalators can be dramatically enhanced by single atomic-layered graphene oxide (GO) sheets. Through π-π stacking interaction mainly between GO and the aromatic ligands of intercalators, the conjugates of GO and a small intercalator could be formed. Because of the large planar structure of the GO sheets, the coupling of GO with the small intercalators increased their affinity to DNA. Owing to the formation of conjugates with GO, the binding site of small intercalators to DNA was also changed from a minor groove to a major groove. Notably, GO and small intercalator conjugates exhibited higher cytotoxicity than that of the small intercalator alone. The results open up potential applications of GO for new chemotherapeutic agents that work through DNA intercalation.