The whisker method using anisole solvent was developed for effective production of high-aspect-ratio poly (3-alkylthiophene) (P3AT) nanofibers, and alkyl chain length dependence on nanofiber formation and their properties were fully investigated. The nanofibers have an anisotropic cross section of 3-4 nm height and 24-27 nm width, which slightly increase with the alkyl chain length, and the aspect ratio reaches 100-1000. The nanofibers consist of more than 104 parallel stacks of the extended polymer backbones along the nanofiber long axis and of 2-3 laminated layers of the polymer backbones separated by alkyl side chains. The nanofiber formation originates from quasi-one-dimensional crystallization of P3ATs induced by both an attractive pi-pi* interaction between polymer backbones and the crystallization of alkyl side chains. Carrier transport properties of a AuCl3-doped nanotiber network and single nanofibers, both of which are explained by a quasi-one-dimensional variable-range hopping (VRH) model irrespective of alkyl chain length, indicate that the origin of the random potential that localizes the carriers should be attributed not to the bridges between nanotibers but to some factor involved in a single nanofiber.