A review of the characterization and functions of extracellular polymeric substances (EPS) of microbial aggregates in biological wastewater treatment systems is presented in this paper. EPS represent the complex high-molecular-weight mixture of polymers excreted by microorganisms generated from cell lysis as well as adsorbed inorganic and organic matter from wastewater. EPS exhibit a three-dimensional, gel-like, highly hydrated matrix that facilitates microbial attachment, embedding, and immobilization. EPS play multiple roles in containments removal, and the main components of EPS crucially influence the properties of microbial aggregates, such as adsorption ability, stability, and formation capacity. Moreover, EPS are important to sludge bioflocculation, settleability, and dewatering properties and could be used as carbon and energy sources in wastewater treatment. However, due to the complex structure of EPS, related knowledge is incomplete, and further research is necessary to understand fully the precise roles in biological treatment processes.