One-part (1K) polyurethane (PU) adhesive has excellent bulk strength and environmental resistance. It is therefore widely used in many fields, such as construction, transportation, and flexible lamination. However, when contacting non-polar polymer materials, the poor adhesion of 1K PU adhesive may not be able to support its outdoor applications. To solve this problem, plasma treatment of the non-polar polymer surface has been utilized to improve adhesion between the polymer and 1K PU adhesive. The detailed mechanisms of adhesion enhancement of the 1K PU adhesive caused by plasma treatment on polymer substrates have not been studied extensively because adhesion is a property of buried interfaces which are difficult to probe. In this study, sum frequency generation (SFG) vibrational spectroscopy was used to investigate the buried PU/polypropylene (PP) interfaces in situ nondestructively. Fourier-transform infrared spectroscopy, the X-ray diffraction technique, and adhesion tests were used as supplemental methods to SFG in the study. The 1K PU adhesive is a moisture-curing adhesive and usually needs several days to be fully cured. Here, time-dependent SFG experiments were conducted to monitor the molecular behaviors at the buried 1K PU adhesive/PP interfaces during the curing process. It was found that the PU adhesives underwent rearrangement during the curing process with functional groups gradually becoming ordered at the interface. Stronger adhesion between the plasma-treated PP substrate and the 1K PU adhesive was observed, which was achieved by the interfacial chemical reactions and a more rigid interface. Annealing the samples increased the reaction speed and enhanced the bulk PU strength with higher crystallinity. In this research, molecular mechanisms of adhesion enhancement of the 1K PU adhesive caused by the plasma treatment on PP and by annealing the PU/PP samples were elucidated.