Low-molecular-weight (LMW, <1000 Da) dissolved organic matter (DOM) plays a significant role in metal/organic pollutant complexation, as well as photochemical/microbiologicalprocesses in freshwater ecosystems. The micro size and high reactivity of LMW-DOM hinder itsprecise characterization. In this study, Suwannee River fulvic acid (SRFA), a commonly used referencematerial for aquatic DOM, was applied to examine the optical features and molecular composition ofLMW-DOM by combining membrane separation, ultraviolet–visible absorption and Orbitrap massspectrometry (MS) characterization. The 100–500 Da molecular weight cut-off (MWCO) membranehad a better performance in regard to separating the tested LMW-DOM relative to the 500–1000 DaMWCO membrane. The ultraviolet–visible absorbance decreased dramatically for the retentates,whereas it increased for the dialysates. Specifically, carbohydrates, lipids and peptides exhibited highselectivity to the 100–500 Da MWCO membrane in early dialysis. Lignins, tannins and condensedaromatic molecules displayed high permeability to the 500–1000 Da MWCO membrane in late dialysis.Overall, the retentates were dominated by aromatic rings and phenolic hydroxyls with high O/Cwa(weighted average of O/C) and low H/Cwa. Conversely, such dialysates had numerous aliphaticchains with high H/Cwa and low O/Cwa compared to SRFA. In particular, LMW-DOM below200 Da was identified by Orbitrap MS. This work provides an operational program for identifyingLMW-DOM based on the SRFA standard and MS analysis.