The adsorption of humic substances on Ag nanoparticles (AgNPs) is of crucial environmental importance and determines the toxicity of these NPs and the structure of adsorbed organic matter. In this work, the adsorption of two standard soil and leonardite International Humic Substances Society humic acids was studied on AgNPs of different sizes, shapes (spherical and star-like), and interfacial chemical compositions. Surface-enhanced optical (Raman and fluorescence) spectroscopies were used to follow the specific chemical groups involved in this adsorption. By means of the latter optical techniques, information regarding the binding mechanism and the macromolecular aggregation can be deduced. The influence of the surface chemical composition induced by the different functionalizations of the interfaces of these NPs is highly important regarding the chemical interactions of these complex organic macromolecules. The surface functionalization with positively charged alkyl diamines led to a large increase in the adsorption as well as a strong structural rearrangement of the macromolecule once adsorbed onto the surface.