The proton binding heterogeneity of forest soilfulvic acid (FA) sub-fractions (FA3-FA13) was investigated by use of synchronous fluorescence spectra (SFS), combined with two-dimensional correlation spectroscopy (2D COS) and modified Stern-Volmer model. XAD-8 adsorption techniques coupled with stepwise elution using Na4P2O7 buffers were successfully developed to separate the soil FA into FA sub-fractions with low heterogeneity (FA3-FA13). FA3-FA13 contained protein-like (i. e., tyrosine-like and tryptophan-like), fulvic-like and humic-like materials, and FA7-FA13 contained more contents of protein-like materials than both FA3 and FA5. The intensities of fluorescence peaks for different fluorescent materials in FA3-FA13 were affected significantly by the change of alkaline pH. The complex distributions of the auto-peaks and cross-peaks in synchronous and asynchronous maps derived from 2D COS were related to the proton binding heterogeneity of fluorescent materials in FA3-FA13. The protein-like, fulvic-like and humic-like materials in FA5-FA13 occurred in the same spectral directionby the pH perturbations. At alkaline pH conditions, the dissociation constants (pKa) of fluorescent materialsin FA3-FA13were quantified by the SFS-2D COS combined with modified Stern-Volmer model. The pKa values of tyrosine-like, tryptophan-like, fulvic-like and humic-like materials were ranged 6.11, 8.93～10.12, 9.32～10.65, and 9.70～10.63 (R>0.854), respectively. The lower pKa value (6.11) of tyrosine-like materials of FA3 indicated that the tyrosine-like material scontained more aromatic structures and adjacent phenolic functional groups. Similar pKa values were presented for the tryptophan-like, fulvic-like and humic-like materials of FA3-FA13(8.93～10.65), and were similar to the pKa values (8.0～12.02) of hydroxyl-benzene and amino acid. This result suggested that the tryptophan-like, fulvic-like and humic-like materials of FA3-FA13 had similar proton affinities, and the phenolic functional groups and amino acid components played a major role in the proton bonding process. The sequential changesof fluorescent materials with specific wavelengths in FA3-FA13 were consistent with their increasing trends of pKa values. Additionally, the heterogeneous distributions of proton binding sites were both presented in different fluorescent materials and same fluorescent materials of FA sub-fractions. The pKa values (6.11～9.16) of tyrosine-like and tryptophan-like materials with specific wavelengths of FA3 were smaller than those of humic-like materials (9.70～9.97), and their increasing trends were consistent with the sequential orders (250 nm→275 nm→425～490 nm). The increasing trends of pKa values of tryptophan-like, fulvic-like and humic-like materials of FA5-FA13 were consistent with the sequential variations: 275 nm (8.93～9.70)→375～495 nm (9.88～10.16)→350 nm (10.65) for both FA5 and FA7, 275 nm (10.11)→290～400 nm (10.35) for FA9, and 265～345 nm (9.32～9.80)→360～450 nm (10.06～10.13) for FA13. In addition, the sequential wavelength changes and pKa values of same fulvic-like materials in FA13 showed the orders of 325 nm (9.32)→375～425 nm (10.06～10.13). With the advantages of reducing theoverlaps of spectra and capturing the sequential wavelength changes, the SFS-2D COS combined with modified Stern-Volmer model will provide supports for exploring complex interactions between dissolved organic matter and contaminants in future studies.