Thiophenylethyne-based compounds have been extensively investigated for use in some molecular electronic devices. Based on thiophene and acetylene units, a series of oligomers and a polymer with different chain lengths were designed and synthesized via Sonogashira reaction in this work. These compounds were characterized by routine measurements to demonstrate that the optical bathochromic shifts and decreasing band gaps were caused by the increasing length of conjugated backbones. Meanwhile, synchrotron-based two-dimensional grazing-incidence X-ray diffraction was adopted to study the molecular stacking orientations with respect to the surfaces. The closer distance between the lamellar layers was shown to contribute to the better conducting property of the compounds. Moreover, the microscopic structural study directly provided further explanation for the good performance of the polymer in this system. Some top-gate field-effect transistors were fabricated via solution processing with the selected oligomers and the polymer. The polymer devices showed good charge transport mobility performance at 0.42 cm² V⁻¹ s⁻¹, with an on–off ratio of 1.59 × 10³, which are much better than those of the oligomers.