Contamination by accidental cutaneous contact with the commercial products and the air pollutants raised a considerable health and safety issue. This study aimed to trace the dynamics of the 20 nm gold nanoparticle (GNP) penetration and accumulation in rat skin tissues using a surface-enhanced Raman scattering (SERS) technique. After the topical application of GNPs on rat skin surface, the SERS spectra were recorded for every 15 μm to an overall depth of 75 μm from skin surface for 150 min. The processes of GNP penetration in rat skin were accompanied by aggregation of GNPs, which affected SERS spectra. The results revealed that 20 nm GNPs can penetrate through stratum corneum layer, viable epidermis layer, and then into dermis layer. This study demonstrated for the first time the potential of SERS spectroscopy to monitor the penetration and accumulation of GNPs in rat skin.