With the aim of understanding the relationships between polymer self-organization and charge carrier mobility of polymer organic field-effect transistor (OFET), we investigate crystalline microstructure change of annealing-induced self-organization of regioregular poly(3-hexylthiophene) (RR-P3HT) active thin layer in polymer OFET by synchrotron radiation grazing incident X-ray diffraction (GIXRD). The crystalline microstructures of RR-P3HT thin film with different preparation methods (spin-coating and drop-casting) and different concentrations (2.5 mg/ml and 3.5 mg/ml) at various annealing temperatures are studied. These results present that, the crystalline structures of RR-P3HT active layers annealed at 150 ℃ are better and enhanced to charge transport, which tend to pack form the thiophene rings are perpendicular and the π-π interchain stacking parallel to the substrate. Furthermore, we find that an appropriate annealing temperature can facilitate the crystal structure of edge-on form, resulting in field-effect mobility enhancement of polymer OFET.