Interaction between buckminsterfullerene and humic substances of different origin was investigated using fluorescence spectrometry as function of concentration of humic substances, pH and ionic strength. Binding constants of humic substances and buckminsterfullerene were calculated. It can be suggested that the complexation was driven by hydrophobic interaction depending on properties of the interacting compounds. Hydrophobic interaction model was indicated by linear Stern-Volmer plot, and high K d values characterized interaction between buckminsterfullerene and humic acids. The results of this study support the development of understanding of fate of nanomaterials in environment as well as development of analytical techniques for nanomaterials in water and wastewater treatment approaches.