Neural stem cell (NSC)-based therapy is a promising candidate for treating neurodegenerative diseases and the preclinical researches call an urgent need for regulating the growth and differentiation of such cells. The recognition that three-dimensional culture has the potential to be a biologically significant system has stimulated an extraordinary impetus for scientific researches in tissue engineering and regenerative medicine. Here, A novel scaffold for culturing NSCs, three-dimensional bacterial cellulose-graphene foam (3D-BC/G), which was prepared via in situ bacterial cellulose interfacial polymerization on the skeleton surface of porous graphene foam has been reported. 3D-BC/G not only supports NSC growth and adhesion, but also maintains NSC stemness and enhances their proliferative capacity. Further phenotypic analysis indicated that 3D-BC/G induces NSCs to selectively differentiate into neurons, forming a neural network in a short amount of time. The scaffold has good biocompatibility with primary cortical neurons enhancing the neuronal network activities. To explore the underlying mechanisms, RNA-Seq analysis to identify genes and signaling pathways was performed and it suggests that 3D-BC/G offers a more promising three-dimensional conductive substrate for NSC research and neural tissue engineering, and the repertoire of gene expression serves as a basis for further studies to better understand NSC biology. Copyright © 2021 Elsevier B.V. All rights reserved.