This study investigated the effects of surface functional groups, cation exchange capacity (CEC), surface charge, sesquioxides and specific surface area (SSA) of three soil clay fractions (SCFs) (kaolinite–illite, smectite and allophane) on the retention of dissolved organic carbon (DOC) in soils. Physico-chemical properties of the SCFs before and after removing native carbon and/or sesquioxides were characterised, and the DOC adsorption–desorption tests were conducted by a batch method. Native organic carbon (OC)/sesquioxide removal treatments led to a small change in the CEC values of kaolinite–illite, but significant changes in those of smectite and allophane. The net negative surface charge increased in all samples with an increase in pH indicating their variable charge characteristics. The removal of native OC resulted in a slight increase in the net positive charge on soil clay surfaces, while sesquioxide removal increased the negative charge. Changes in the functional groups on the SCF surfaces contributed to the changes in CEC and zeta potential values. There was a strong relationship (R² = 0.93, p < 0.05) between the Langmuir maximum DOC adsorption capacity (Q_max) and SSA. The Q_max value also showed a moderately strong relationship (R² = 0.55, p < 0.05) with zeta potential (at pH 7). Q_max was only poorly correlated with CEC and native OC content. Therefore, along with SSA, the surface charge and functional groups of SCFs played the key role in determining the adsorption affinity and hence retention of DOC in soils.