Dissolved organic matter (DOM) plays a substantial role in wastewater treatment systems. Fluorescence is an important property of DOM and its use is promising for DOM characterization, but has rarely been extended to probing the basic physicochemical properties such as hydrophobicity and molecular weight. This study explores the possible linkages between the fluorescence properties and hydrophobicity/molecular weight of DOM, through case studies from three wastewater treatment plants (two membrane bioreactors and one oxidation ditch). The fluorescence properties of different hydrophobic/hydrophilic and molecular-weight fractions of DOM were obtained using excitation-emission matrix (EEM) spectroscopy and size-exclusion chromatography with fluorescence detection. The EEM spectra were interpreted using techniques of fluorescence regional integration, parallel factor analysis, fluorescence spectroscopic indices, and a novel energetic mapping based on fluorophore energy levels. It was found that for all the three plants, the hydrophobic fractions of DOM had a higher fluorescence intensity per UV absorbance (indicating a higher quantum yield) as well as a larger Stokes shifts than the hydrophilic fraction. The lower-molecular-weight fractions generally exhibited a higher fluorescence intensity per total organic carbon (indicating a higher fluorophore density), with the fluorescence distribution at slightly smaller excitation and emission wavelengths. These phenomena were explained via analysis of the fluorophore energy state during the excitation/emission process. The scale of the π-conjugated system in DOM molecules may serve as an intermediate factor in the correlations between the hydrophobicity/molecular weight and the fluorescence properties. These correlations may assist in developing fluorescent probes for the DOM characteristics during the process monitoring of wastewater treatment plants.