Electroactive nanofibers were fabricated by in situ polymerization of aniline on the surface of tobacco mosaic virus (TMV) using sodium poly(styrenesulfonate) (PSS) as dopant. These electroactive TMV/PANi/PSS nanofibers were employed to support growth of neuronal cells, resulting in augmentation of the length of neurites. In addition, the percentage of cells with neurites was increased in comparison to cells cultured on TMV-derived nonconductive nanofibers. The TMV-based electroactive nanofibers could be aligned in capillaries that could guide the outgrowth direction of neurites, increase the percentage of cells with neurites, and lead to a bipolar cellular morphology. Our results demonstrate that the electroactivity and topographical cues provided by TMV/PANi/PSS nanofibers can synergistically stimulate neural cells differentiation and neurites outgrowth, which make it a promising scaffolding material for neural tissue engineering.