Dissolved organic matter (DOM) in aquatic systems is highly heterogeneous in molecular size and chemical composition, as characterized by the flow field flow fractionation (FlFFF) and other analytical techniques capable of simultaneous size-separation and chemical characterization. In addition, the fate, transport, and biological/chemical reactivity of natural DOM along the aquatic continuum are largely related to its molecular size and composition. Over the past decade, the fluorescence excitation-emission matrices (EEMs) coupled with parallel factor analysis (PARAFAC) have been widely used for the characterization of fluorescent DOM in natural waters, usually for a large set of samples to be statistically significant. However, characterization of molecular-size dependent chemical properties of DOM using the EEMs-PARAFAC technique remains challenging, especially for a single DOM sample.  Here we present a newly-developed method coupling the FlFFF size fractionation with EEMs and PARAFAC analysis to elucidate the continuous changes in fluorescent DOM composition with molecular size from 0.3-kDa to 700-nm in a single DOM sample. Based on the analysis of a single riverwater sample, results from FlFFF-EEMs coupling show that the humic-like components, including Peaks A and C, were mostly partitioned within the 3-100 kDa size fractions, while the protein-like components, Peaks B and T, were dominantly present in the >100 kDa size fractions.  PARAFAC analysis on EEM spectra of different size fractions in the sample fractionated by FlFFF identified three humic-like fluorescent components (C1, C2 and C3), all presented mostly in the 0.3-20 kDa size ranges, and one protein-like component (C4) mostly in the molecular size range from ~30 kDa to >1000 kDa with increasing relative abundance with molecular size. The change in component ratios (C₄/C_1-3) with molecular size exhibited a bimodal distribution with high values in both the <0.3 kDa and >20 kDa size fractions, similar to the size distribution pattern observed for the fluorescence index ratios between biological index and humification index (BIX/HIX). These results indicated that protein-like DOM components were mostly autochthonous in nature and partitioned either in lower or higher molecular size ranges, while humic-like DOM components were mostly in the molecular size range of 0.3-10 kDa in the sample.  New results derived from the FlFFF-EEM-PARAFAC coupling technique provide a holistic understanding of the size-dependent heterogeneity of DOM in a single sample and can be used to reveal the dynamic changes of DOM molecular size and composition in the aquatic continuum across the river-lake and river-sea interfaces.