The interaction of biomedical imaging gold nanoparticles with environmental entities like humic substances was investigated applying UV-Vis absorbance and fluorescence spectroscopy. Gold nanoparticles have potential applications in drug delivery and biomedical imaging; however their fate in the environment is relatively unknown. The interactions of gold nanoparticles with environmental entities like humic substances present in natural aquatic systems will play a vital role in their fate and transformation. Humic substances (HS) are fluorescent in nature, giving us an interesting tool to characterize the interaction between these natural polyelectrolytes and model water soluble gold nanoparticles. When gold nanoparticles were exposed to HS, the fluorescence map (excitation/emission/intensity) of the humic substances was observed to be quenched. This quenching exhibited concentration dependent behavior. The conclusion was that the gold nanoparticles are associating with these water soluble polyelectrolytes closely enough that energy could be transferred between the different species. However, no precipitation was observed, indicating that gold nanoparticles might be mobilized in surface waters due to such interactions. The aggregation behavior of the gold nanoparticles/HS was also studied using UV-Vis absorbance. Absorbance of the gold nanoparticles was observed at 519 nm in the control and this response shifted to longer wavelengths (650-800 nm) as the concentration of HS increased, indicating that the gold nanoparticles in the presence of humic substances were aggregating. This behavior is best observed as a difference in the spectra of the gold nanoparticles/HS and the spectra of the HS alone.