Poly(3-alkylthiophene)s (P3AT) are among the most widely used conjugated polymers for applications in polymer solar cells and thin-film transistors. In this study, the influence of the casting solvent on the conformational structure and fluorescence properties of the isolated chains of a regioregular and a regiorandom poly(3-hexylthiophene) (denoted by rr-P3HT and ra-P3HT, respectively) has been systematically investigated by single molecule spectroscopy. The isolated chains were dispersed in polystyrene matrix by casting from their ultraclilute solutions with tetrahydrofuran (THF) or toluene. ra-P3HT chains were found to contain mostly high-energy emitting species with short conjugating length, while the low-energy emitting sites existed predominantly in rr-P3HT chains irrespective of the casting solvent. The measurement of the modulation depth revealed that rr-P3HT chains cast from THF formed defect cylinder structure. The regioregular chains cast from toluene exhibited relatively regular folding to form rod structure as a result of the amplification of the segmental attraction in the poorer solvent. The casting solvent had insignificant effect on the conformational structure of ra-P3HT, where the isolated chains predominantly showed defect coil conformation irrespective of the casting solvent.