We investigated the influence of phenolic OH groups on the complexation behavior of humic acid (HA) with UO₂²⁺ions at pH 4. Starting from synthetic HA type M1, natural HA Aldrich, and natural HA Kranichsee, we synthesized modified HAs with blocked phenolic OH groups by derivatization with diazomethane. The partial blocking of phenolic OH groups was confirmed by a radiometric method which quantitatively determined the functional groups and by FTIR spectroscopy. The complexation behavior of the chemically modified and unmodified HAs with UO₂²⁺ions was investigated by time-resolved laser-induced fluorescence spectroscopy. The experimental data were evaluated with the metal ion charge neutralization model. We determined comparable complexation constants for all HAs. Two modified HAs (type M1 and Aldrich) had significantly lower loading capacities for UO₂²⁺ions (10.5 ± 0.9% and 9.7 ± 1.6%, respectively) than the corresponding unmodified HAs 18.0 ± 2.0% and 17.5 ± 1.6%, respectively). This indicates that the blocking of the phenolic OH groups changes the complexation behavior of HAs.