With all advantages and desirable characteristics of organic polymers as potential candidate for the next generation of electronics, optimizing the performance of plastic electronics still remains a great challenge. This is primarily because optoelectronic properties of polymer aggregates are a complex function of their molecular packing structure. Crystalline, self-assembled, two-dimensional nanofibers of P3HT polymers represent an attractive platform for studying optical and electronic properties of exciton coupling due to their nominal (highly crystalline) internal chain packing structure. We have combined single molecule spectroscopy methods with scanning probe microscopy techniques (AFM, KPFM) and transmission electron microscopy (TEM) to optically assess the exciton coupling type and strength in individual nanofibers as a function of polymer chain length and examine their electronic properties. Correlating the optical and electrical properties with nanofibers’ width unravels the impact of chain conformation on the strength, effective dimensionality, and dynamics of exciton coupling as well as on HOMO energy level. It also indicates the impact of confinement on the molecular packing and thus optoelectronic properties. This provides guidelines for effective tuning of the HOMO energy level and dimensionality of exciton coupling in P3HT nanofibers through modulating chain conformation.