Extracellular polymeric substances (EPS) were considered as fundamental substances in colony formation; however, the understanding of EPS composition remains limited. This study analyzed the content and composition of EPS fractions (soluble EPS, loosely bound EPS, and tightly bound EPS) of four Microcystis species from laboratory cultures in both unicellular and colonial morphologies, as well as colonies collected during Microcystis blooms, using fluorescence excitation - emission matrix spectroscopy combined with parallel factor analysis (EEM-PARAFAC). This method enables to make insight into protein-like and humic acid-like components but cannot detect polysaccharides. The EPS was successfully categorized into three humic acid-like components (C1 – C3) and a protein-like component (C4). Component C1 was discovered to be involved in colony formation and colony size growth of Microcystis. EPS content varied among Microcystis morphospecies, such as M. aeruginosa, M. wesenbergii and M. ichthyoblabe, and this was significantly affected by the environmental constraints rather than the morphospecies. The proportion of C1 relating to larger colony size was negatively correlated to temperature and concentrations of TN and TP. The tightly bound EPS directly promoted colony formation, but the soluble EPS or loosely bound EPS alone did not induce colony formation in Microcystis. These results advanced the current knowledge on the chemical materials involved in the colony formation of Microcystis and provided new clues in unicellular-multicellular transformation as well as colonial morphology changes in Microcystis.