Using three analytical techniques of size exclusion chromatography (SEC), fluorescence excitation-emission matrix (EEM), and dissolved organic nitrogen (DON) measurement, differentiating characteristics of effluent organic matter (EfOM) from natural organic matter (NOM) have been investigated. SEC reveals a wide range of molecular weight (MW) for EfOM and high amount of high MW polysaccharides, and low MW organic acids compared to NOM. Clear protein-like peaks using fluorescence EEM were a major feature of EfOM distinguishing it from NOM. Fluorescence index (FI), an indicator to distinguish autochthonous origin from allochthonous origin, differentiated EfOM from NOM by exhibiting higher values, indicating a microbial origin. In EfOM samples, DON present in higher amounts than NOM.