In this study, a two-site fluorescence quenching data treatment was developed based on the Ryan–Weber model to investigate natural organic matter complexation with uranyl ion. Parameters for each ligand site including stability constant (log K), ligand site concentration (CL) and residual fluorescence intensity (Ires) were determined using this approach. Parallel factor (PARAFAC) analysis was used to obtain quantitative data for peaks from fluorescence excitation emission matrix (EEM) spectra. This model was tested using a Cu2+ titration with a mixed system of tyrosine and tryptophan to simulate two ligands. Relatively good agreements with literature values (within 3.29% for log K values) indicated the validity of the model. The model was further applied in the complexation study between soil fulvic acid (SFA) and UO22+ since two components were predicted using core consistency diagnoses (CORCONDIA) for our SFA sample. Results for 20mg/L SFA at pH3.50 for the peak at λex=330nm, λem=450nm were log K=4.49, CL=5.05μmol/L, and Ires=15.1 and for the peak at λex=240nm, λem=450nm were log K=4.56, CL=4.56μmol/L, and Ires=1.2.