Poly(3-hexylthiophene) (P3HT) nanofibers with different nanostructures and intrachain order were prepared, and the interactions between different P3HT nanofibers and GO were investigated by UV-vis, Raman, and atomic force microscopy (AFM) measurements, etc. We found that at the liquid-GO interface the conjugated length of nanofibers with lower intrachain order increased since polymer chains fractions were more likely to be involved in the formation of the nanofibers. We also demonstrated that GO can be differently doped when interacting with different P3HT nanofibers, which were confirmed by Raman spectroscopy and AFM. Kelvin probe force microscopy (KPFM) was also used to monitor the surface potential (SP) change of P3HT nanofibers after they interacted with GO and further confirmed the varied interactions between different P3HT nanofibers and GO.