This research investigated the effect of a high-voltage external electric field on the ordered structure of molecular chains and hole mobility in regioregular poly(3-hexylthiophene) (P3HT) with different molecular weights through X-ray diffraction, atomic force microscopy, transmission electron microscopy, Fourier transform infrared spectroscopy, micro-Raman spectroscopy, UV-vis spectroscopy, photoluminescence spectroscopy, and organic field-effect transistors. The optimal magnitude of the external electric field was 5000 V/cm. With the optimized electric field applied to a series of P3HT films, the carrier mobility of all P3HT films increased, and the increase rate changed from 105% to 56%, closely depending on the increase in molecular weight from 33 kg/mol to 100 kg/mol. The results indicated that the increase in carrier mobility was attributed to the P3HT conformation order, which was controlled by the external electric field. Molecular weight was a critical factor in determining the P3HT conformation response to the external electric field. The external electric field orientated lower-molecular-weight (33 kg/mol) P3HT into ordered structures more obviously than higher-molecular-weight (100 kg/mol) P3HT. This research contributes to the understanding of the effect of an external electric field on the ordered structure of the chains and carrier mobility in P3HT with different molecular weights. This research also reveals the regularity and mechanism of the formation of ordered structures and essentially enhances the carrier mobility of P3HT films with different molecular weights, to fabricate photovoltaic devices with high efficiency, based on polymer physics.