In recent years organic field-effect transistors have received extensive attention, however, it is still a great challenge to fabricate monolayer-based devices of conjugated polymers. In this study, one single layer of poly(2,5-bis(3-alkylthiophen-2-yl)thieno[3,2-b]thiophene) is directly dip-coated, and its self-assembly is precisely tuned from nanofibers to granular aggregates by controlling the dielectric roughness on a sub-nanometer scale. The charge carrier transport of the monolayer transistor exhibits a strong dependence on the dielectric roughness, which is attributed to the roughness-induced effects of higher densities of grain boundaries and charge trapping sites as well as surface scattering. These results mark a great advance in the bottom-up fabrication of organic electronics.