In biological wastewater treatment systems, extracellular polymeric substances (EPS) are continuously excreted as a response to environmental changes and substrate conditions. It could severely affect the treatment efficacy such as membrane fouling, dewaterability and the formation of carcinogenic disinfection by-products (DBPs). The heterogeneous dissolved organic matter (DOM) with varying size and chemical nature constitute a primary proportion of EPS. In the last few decades, fluorescence spectroscopy has received increasing attention for characterizing these organic substances due to the attractive features of this low-cost spectroscopic approach, including easy sample handling, rapid, non-destructive and highly sensitive nature. In this review, we summarize the application of fluorescence spectroscopy for characterizing EPS and provide the potential implications for online monitoring of water quality along with its limitations. We also link the dynamics of fluorescent dissolved organic matter (FDOM) in EPS with operational and environmental changes in wastewater treatment systems as well as their associations with metal binding, membrane fouling, adsorption, toxicity, and dewaterability. The multiple modes of exploration of fluorescence spectra, such as synchronous spectra with or without coupling with two-dimensional correlation spectroscopy (2D-COS), excitation-emission matrix (EEM) deconvoluted fluorescence regional integration (FRI), and parallel factor analysis (PARAFAC) are also discussed. The potential fluorescence indicators to depict the composition and bulk characteristics of EPS are also of interest. Further studies are highly recommended to expand the application of fluorescence spectroscopy paired with appropriate supplementary techniques to fully unravel the underlying mechanisms associated with EPS.