Phenanthrene (Phe) is a toxin and is ubiquitous in the environment. The sediment humic substances (HS) that bind Phe affect the fate, transport, degradation, and ecotoxicology of Phe. This study investigated Phe sorption constants on size-fractioned HS extracted from river sediment. Fractions were identified as HHS (10 kDa to 0.45 µm), MHS (1-10 kDa), and LHS (<1 kDa). A fluorescence quenching (FQ) method was used to determine the Phe log K HS on size-fractioned HS; the values ranged from 3.97 to 4.68 L/kg-C. The sorption constant (log K HS ) is a surrogate of the binding capacity between HS and Phe, where a high log K HS reduces the toxicity and degradation of Phe. The log K HS values on HHS and MHS were significantly higher than the values on LHS (p = 0.015). The SUVA 254 values of HHS and MHS were also significantly higher than the LHS value (p = 0.047), while fluorescence index (FI) and S 275-295 values were significantly lower than the LHS values (p < 0.005). The HHS and MHS had a higher aromaticity and more terrestrial sources than LHS. The log K HS had a significant correlation with the selected optical indicators (p < 0.002), which suggested that the HS-bound Phe was positively affected by high aromaticity, terrestrial sources, and HS molecular weight. The results demonstrated that optical methods successfully obtained log K HS and the chemical properties of fractioned HS as well as the influenced factors of log K HS . Moreover, even the LHS had a capacity to bind with Phe.