A facile approach was developed to fabricate patterned substrates of nano-graphene oxide, demonstrating highly localized and efficient gene delivery to multiple cell lines in a substrate-mediated manner. The GO substrates served as a valid platform to preconcentrate PEI/pDNA complexes and maintain their gradual releasing for a relatively long period of time. Our approach allowed successful gene delivery in selected groups of cells on the stripe-patterned GO substrates, without transfecting their neighbor cells directly cultured on glass. These GO substrates exhibited excellent biocompatibility and enabled effective gene transfection for various cell lines including stem cells, thus promising important applications in stem cell research and tissue engineering.