Abstract: It has been realized that condensed structure control over poly(3-alkylthiophenes) (P3ATs) at nanoscale is essential for the improvement of the properties of optoelectronic devices. The aggregation of P3ATs at nanoscale driven by interchain π-π interactions typically shows the one-dimensional nanowires (NWs) morphology. Poly(3-dodecylthiophene) (P3DDT) has good solubility in normal organic solvents. This characteristic makes the preparation of P3DDT nanofibers/wires relatively difficult and out of control. In order to improve the crystallinity of P3DDT in solvents, P3DDT nanofibers were successfully prepared by low temperature annealing and self-nucleation method in this work. The influences of self-nucleation temperature on the crystallinity and crystal modification of P3DDT nanofibers were investigated. The results show that the homogeneous and high yield nanofibers can be obtained after the P3DDT solution is annealed at low temperature and then crystallized at room temperature. On this basis, the additional self-nucleation step can further improve the crystallinity of nanofibers. The results of infrared spectra show that the samples obtained after low temperature annealing and self-nucleation result in Form Ⅱ modification rather than the typical Form Ⅰ. Temperature dependent infrared test further reveals the transformation of P3DDT nanofibers from Form Ⅱ crystal to Form Ⅰ, and then the phase transition in the subsequent melting. This work is expected to shed light on the preparation and polymorphic control of poly(3-alkylthiophene) (P3ATs) nanofibers/wires. The investigation results will benefit for the understanding of the crystallization behavior of conductive polymers, providing the theoretical guideline for the structural control of the P3ATs NWs as well as the optimization of the properties of the devices in industrial process.