Sludge aggregation and biofilm formation are the most effective approaches to solve the washout of anammox microorganisms. In this study, the structure and composition of EPS (extracellular polymeric substances) were investigated to elucidate the factors for the anammox aggregation property. Anammox sludge taken from 18 lab-scale and pilot-scale reactors treating different types of wastewater was analyzed using EEM-PARAFAC (excitation-emission matrix and parallel factor analysis), FTIR (Fourier transform infrared spectroscopy) and real-time PCR combined with multivariate statistical analysis. The results showed that slime and TB-EPS (tightly bound EPS) were closely related with water quality and sludge morphology, and could be used as the indicators for anammox microbial survival ability and microbial aggregate morphology. Furthermore, slime secreted from anammox bacterial cells may be exhibited higher viscosity to the sludge surface and easily formed the gel network to aggregate. Large amounts of hydrophobic groups of protein in TB-EPS promoted the microbial aggregation. The mechanisms of anammox aggregation explored in this study enhanced the understanding of anammox stability in wastewater treatment processes.