Despite extensive studies on the archetypal poly(3-hexylthiophene-2,5-diyl) (P3HT) over the past decade, a concurrent investigation of the surface roughness influence on polymorphism, density of states (DOS), photoluminescence (PL), and hole mobility is missing. We report on the effect of substrate roughness on the optical and electrical properties of P3HT thin films deposited on smooth c-Si (roughness < 1 nm) and rough ITO (roughness ∼ 6 nm) substrates. Grazing-incidence wide-angle X-ray scattering and the energy-resolved electrochemical impedance spectroscopy were used to correlate the two polymorphs with the defect DOS in P3HT thin film grown on ITO substrate. The Jablonski diagram reconstructed from PL spectra elucidates the PL bleaching associated with sub-bandgap defect states above the highest occupied molecular orbital found in the case of the rough ITO substrate. A decrease in the PL intensity of the 0–1 transition caused by this extra band of defect states is presumably due to a non-radiative transition from the excited to the ground state or reduction of transition probability. The observed sub-band gap states have no effect on hole mobility. However, the continuum of gap states, which increases with decreasing layer thickness on both substrates, lowers the hole mobility by one order.