In recent years, the utilization of nanomaterials such as carbon nanotubes (CNTs) in the field of neuroscience has forever changed the approach to nerve-related research. The array of novel properties CNTs possess allows them to interact with neurons at the nanodimensional scale. In this study, a CNT rope substrate is developed to allow the electrical stimulation of neural stem cells (NSCs) in culture medium and the in situ observation of the response of these stem cells after stimulation. CNTs are synthesized by chemical vapor deposition and prepared into a ropelike structure with a diameter of 1 mm and length of 1.5 cm. NSCs are differentiated on the CNT rope substrate while the direction of neurite outgrowth, phenotype, and maturity of the NSCs are analyzed. Fluorescence and scanning electron microscopy demonstrate that neurite extension favors the direction of the spiral topography on the CNT rope. NSCs plated on CNT ropes are boosted towards differentiated neurons in the early culture stage when compared to conventional tissue culture plates via the analysis of neuronal gene and protein expressions by quantitative polymerase chain reaction and immunostaining, respectively. Furthermore, a set of electrical stimulation parameters (5 mV, 0.5 mA, 25 ms intermittent stimulation) promotes neuronal maturity while also increasing the speed of neurite outgrowth. These results indicate that an electroconductive CNT rope substrate along with electrical stimulation may have a synergistic effect on promoting neurite elongation and boosting effects on the differentiation of NSCs into mature neuronal cells for therapeutic application in neural regeneration.