© 2019 American Chemical Society. In this work, we study the interaction of a graphene oxide (GO) substrate and adsorbed rhodamine B (RhB) molecules by Raman spectral deconvoluted scanning. We achieved the deconvolution of a large number of spectra with overlapping Raman bands of GO and RhB. The evolution of the deconvolution parameters is shown as two-dimensional (2D) spatial maps, cross-correlation graphs, and evolution curves of the statistical representation derived from the data histogram. We show that at resonant excitation of the RhB molecules, the dye fluorescence is quenched, and the GO Raman bands are modified. We report a downshift in the G-band position and an increase in the intensity of the defect-activated D- and D′-band with a higher amount of adsorbed RhB molecules. These results will be discussed considering previously reported works on Raman spectra dependence with molecular-doped and gate-voltage-doped graphene.