Neurodegenerative diseases mainly affect the vital characteristics of neurons, including axons. The fabricated nanotopographies can cause axonal regeneration manipulating the migration and differentiation of cells. Here, we present a protocol for the fabrication of nanosubstrate incorporated with nanogroove topography with a coated layer of polyaniline-chitosan (PANI-C) nanocomposite. We describe steps for investigating differences between bulk polydimethylsiloxane (PDMS) sheets and embedding sheets with nanogrooves. We then detail procedures for coating with a PANI-C nanocomposite layer on the substrate. For complete details on the use and execution of this protocol, please refer to Afsharian et al. 1 .