Optic neuropathies, such as glaucoma and Leber's hereditary optic neuropathy (LHON) lead to retinal ganglion cell (RGC) loss and therefore motivate the application of transplantation technique into disease therapy. However, it is a challenge to direct the transplanted optic nerve axons to the correct location of the retina. The use of appropriate scaffold can promote the proper axon growth. Recently, biocompatible materials have been integrated into the medical field, such as tissue engineering and reconstruction of damaged tissues or organs. We, herein, utilized nano-imprinting to create a scaffold mimicking the in vitro tissue microarchitecture, and guiding the axonal growth and orientation of the RGCs. We observed that the robust, long, and organized axons of human induced pluripotent stem cell (iPSC)-derived RGCs projected axially along the scaffold grooves. The RGCs grown on the scaffold expressed the specific neuronal biomarkers indicating their proper functionality. Thus, based on our in vitro culture system, this device can be useful for the neurophysiological analysis and transplantation for ophthalmic neuropathy treatment.