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Hyperfine interaction (HFI), originating from the coupling between spins of charge carriers and nuclei, has been demonstrated to strongly influence the spin dynamics of localized charges in organic semiconductors. Nevertheless, the role of charge localization on the HFI strength in organic thin films has not yet been experimentally investigated. In this study, the statistical relation hypothesis that the effective HFI of holes in regioregular poly(3-hexylthiophene) (P3HT) is proportional to 1/N0.5 has been examined, where N is the number of the random nuclear spins within the envelope of the hole wave function. First, by studying magnetoconductance in hole-only devices made by isotope-labeled P3HT we verify that HFI is indeed the dominant spin interaction in P3HT. Second, assuming that holes delocalize fully over the P3HT polycrystalline domain, the strength of HFI is experimentally demonstrated to be proportional to 1/N0.52 in excellent agreement with the statistical relation. Third, the HFI of electrons in P3HT is about 3 times stronger than that of holes due to the stronger localization of the electrons. Finally, the effective HFI in organic light emitting diodes is found to be a superposition of effective electron and hole HFI. Such a statistical relation may be generally applied to other semiconducting polymers. This Letter may provide great benefits for organic optoelectronics, chemical reaction kinetics, and magnetoreception in biology. [ABSTRACT FROM AUTHOR]