The binding of europium to dissolved humic substances (HSs), isolated from deep groundwater in the Horonobe area, Hokkaido, Japan, was evaluated by means of a three-dimensional fluorescence quenching technique. The Ryan and Weber model, extended by the charge neutralization model, was applied to the fluorescence quenching profiles to evaluate the conditional binding constants (K) at pH 5.0 and an ionic strength of 0.1. The K values for fulvic (FA) and humic acids (HA) in the Horonobe groundwater were comparable with each other regardless of the position of the fluorescence peaks. As compared with HSs from Lake Biwa, Aldrich, and Dando soil, the K values of the Horonobe groundwater HSs were the smallest, indicating that the Horonobe groundwater HSs have a lower affinity for the binding of europium compared to the HSs from surface environments. In addition, the results of the Aldrich-based comparison indicate that the Horonobe groundwater HSs have a lower binding affinity for trivalent lanthanides/actinides compared with other groundwater HSs obtained from different aquifer. These findings demonstrate that the characteristics and origin of deep groundwater HSs needs to be taken into consideration assessing the effects of HSs on the speciation of radionuclides in deep groundwater in geological disposal systems.