► FTIR and 3D-EEM are used to analyse the main components of EPS in aerobic granule. ► Results indicate the importance of aromatic protein-like substances in the stable granular sludge. ► The isomers of carbohydrates (1110–1047cm−1) can be attributed to the aerobic sludge granulation. In recent years, lots of the extracellular polymeric substances (EPS) related researches have focused on its role in the granulation and structural stability of aerobic sludge. Three-dimensional fluorescence spectrum (3D-EEM) and fourier transform infrared spectroscopy (FTIR) technologies were used to analyse the main components of sludge EPS during aerobic sludge granulation in this study. Results showed that the components of sludge EPS tended to be stable during aerobic sludge granulation. The peak F (Ex/Em=230/308.5) from 3D-EEM and the predominant spectral band at approximately 1517cm−1 from the FTIR spectra of the matured granular sludge indicated the importance of aromatic protein-like substances together, especially tyrosine in maintaining the stable structure of the granular sludge. Furthermore, the differences in the occurrence position and frequency of C–O bonds (1110–1047cm−1) observed during aerobic sludge granulation showed that the transformations between the isomers and other forms of carbohydrates may be attributed to the formation of aerobic granule.