Reversed-phase high-performance liquid chromatography (RP-HPLC) with online absorbance and fluorescence detections was used for the evaluation of Suwannee River natural organic matter (SRNOM) and its fractions A, B, and C+D, obtained by conventional size exclusion chromatography–polyacrylamide gel electrophoresis (SEC-PAGE) setup, for which the electrophoretic mobility (EM) and the absorptivity varied in the order C+D > B > A, and the molecular size (MS) in the opposite order. Analysis of SRNOM and its fractions in part of their relative irreversible adsorption on C18-column and relative distribution of eluted from the column matter on hydrophobic and hydrophilic peaks showed that hydrophobicity of fractions decreased in order: A > B > C+D. The online fluorescence detection showed that SRNOM and its fractions contained at least three groups of humic substances (HS)-like fluorophores with emission maxima at 435, 455–465, and 455/420 nm and two protein-like fluorophores with emission maxima at around 300 and 340 nm. The HS-like fluorophore with emission maximum at 435 nm was located in the hydrophilic peak in all the samples. Those with maxima at 455–465 nm were detected in hydrophobic peaks of fractions A and B. SEC-PAGE setup followed by RP-HPLC allowed us to develop new approach of SRNOM separation on less heterogeneous compounds mixture for their further study and structural identification.