Surface-enhanced Raman spectroscopy (SERS) has been recognized as a promising analytical technique owing to its merit of nondestructive and fast detection capabilities. However, SERS usually suffers signal interferences from different analytes or a complicated matrix. Separation is an effective approach to solve the signal interference in the application of SERS. It was proposed that two concentric coffee rings could serve as a simple separation platform; however, there are still many questions to be answered for in-depth understanding. In this study, critical parameters during the formation of two concentric coffee rings are characterized for a better understanding of this phenomenon, including surface tension, surface morphology, and surface energy. Two arsenicals, including arsenate (As-V) and cacodylic acid (DMA(V)), are chosen to study the arsenicals' separation/migration mechanism due to their significant difference in chemical properties. In the typical coffee ring, these two arsenicals have signal interference and only DMA(V) is detected via SERS; however, they are detected along the radius of the two concentric coffee rings. The distribution of arsenicals on the two concentric coffee rings is further verified by the chromatographic method. Under this simple platform, interactions between the arsenicals and the surface of the silver nanofilm are pivotal to their migration/separation. By surface modification of silver nanofilm with small molecules, the surface polarity and surface potential are manipulated. The signal dynamics of these two arsenicals are studied on these modified silver nanofilms. It is clear that the electrostatic interaction plays a more important role than the polarity in the arsenicals' migration. This study reveals the mechanism of small molecule migration/separation in the two concentric coffee rings and provides insights for future study of employing this simple platform.