Abstract: Three kinds of substituted polythiophenes,3-hexyl polythiophene (P3HT), 3,4-dipentyl polythiophene (P34PT) and 3-octoxyl polythiophene (P3OOT) were synthesized. The 1H-NMR spectra, UV-Vis absorption spectra and the fluorescence spectra of each polymer are also reported. Theoretical studies on the thiophene with no substituent, 3-ethyl thiophene, 3,4-diethyl thiophene and 3-ethoxyl thiophene dimers were performed with density functional theory (DFT) using Gaussian03 software package. The influences of the substituents on the electrical properties and conformations of polythiophenes are studied and the theoretical results are compared with the experimental results. The energy gaps of polymers turn narrower with the increase of polymerization degrees. The energy gaps and repeating unit lengths of nonsubstituted thiophene, 3-ethyl thiophene, 3,4-diethyl thiophene and 3-ethoxyl thiophene dimers are 4.216 eV, 0.392 7 nm, 4.733 eV, 0.393 9 nm, 3.890 eV, 0.390 8 nm, 5.168 eV, 0.392 5 nm, respectively.