Dewetting of a polymer thin film is directly correlated to the interface with the substrate. We thus combined sum frequency generation (SFG) vibrational spectroscopy and laser scanning confocal microscopy (LSCM) to trace both the interfacial molecular structural and macroscopic morphological evolutions of the polystyrene (PS) thin film on top of the sapphire substrate upon annealing until the dewetting occurred. To reveal the molecular structure of the irreversibly adsorbed layer over the annealing time, a solvent-leaching method was adopted to uncover it from the polymer thin film on the substrate. Experimental results demonstrate the local structural evolution of the irreversibly adsorbed layers, including replacement of adsorption sites (train sequences) from different chains to those from single chains and local structural relaxation of the train sequences, significantly promoted the dewetting. The dewetting of the PS thin film was actually accompanied by such structural evolution of the irreversibly adsorbed layer. We believe this experimental investigation on highlighting the irreversibly adsorbed layer can provide the molecular level insight into how the macroscopic dewetting occurs for a polymer thin film on a substrate.