Poly-3-hexylthiophene (P3HT) nanofibers are 1D crystalline structures with semiconductor properties. When P3HT nanofibers are dispersed in nonconducting solvent, they react to external alternate electric field by aligning along the field lines. This can be used to create layers of ordered nanofibers and is referred to as alternating current poling method. P3HT nanofibers with three different size distributions are fabricated, using self-assembly mechanism in marginal solvents, and used for the alignment studies. Anisotropic absorption of oriented 2 μm long nanofibers exponentially increases with the magnitude of applied field to a certain asymptotic limit at 0.8 V μm-1, while 100-500 nm long nanofibers respond to electric field negligibly. Effective optical birefringence of oriented 2 μm long nanofibers is calculated, based on the phase shift at 633 nm and the average layer thickness, to be 0.41. These results combined with further studies on real-time control over orientation of P3HT nanofibers in liquid solution or host system are promising in terms of exploiting them in electroabsorptive and electrorefractive applications.