Neural stem cells (NSCs), as a self-renewing and multipotent cell population, have been widely studied for never regeneration. Engineering scaffold is one of the important factors to regulate NSCs proliferation and differentiation towards the formation of the desired cells and tissues. Because neural cells are electro-active ones, a conductive scaffold is required to provide three-dimensional cell growth microenvironments and appropriate synergistic cell guidance cues. In this study, a poly (3,4‑ethylenedioxythiophene)/chitosan/gelatin (PEDOT/Cs/Gel) scaffold was prepared via in situ interfacial polymerization, with a nanostructured layer of PEDOT assembling on the channel surface of porous Cs/Gel scaffold. This electrically conductive, three-dimensional, porous and biodegradable PEDOT/Cs/Gel scaffold was used as a novel scaffold for NSCs three-dimension (3D) culture in vitro. It was found that the layer of PEDOT on the channel surface of Cs/Gel scaffolds could greatly promote NSCs adhesion and proliferation. Additionally, under the differentiation condition, the protein and gene analysis suggested that PEDOT/Cs/Gel scaffolds could significantly enhance the NSCs differentiation towards neurons and astrocytes with the up-regulation of β tubulin-III and GFAP expression. In conclusion, these results demonstrated that the PEDOT/Cs/Gel scaffolds as an electrically conductive scaffold could not only promote NSCs adhesion and proliferation but also enhance NSCs differentiation into neurons and astrocytes with higher protein and gene expression. PEDOT-assembled Cs/Gel scaffold will be a promising conductive substrate for NSCs research and neural tissue engineering. Copyright © 2018 Elsevier B.V. All rights reserved.