Heavy metals emission from mining, industrial activities and municipal sewage discharge in water bodies has become a serious environmental concern around the world, posing severe risks to human health and agroecosystem because of their persistence in the environment. Therefore, it is essential to seek effective methods to remediate heavy metal pollution. Compared to conventional methods such as precipitation, adsorption, ion exchange, membrane filtration, biosorption is regarded as an efficient and cost-effective approach to remove metal ions from the environment. Extracellular polymeric substances (EPS) are high molecular weight products secreted by microorganisms (such as algae, bacteria, etc.) and they are typically composed of polysaccharides, proteins, nucleic acids and humic substances. It exhibits a dynamic double-layered structure consisting of soluble-EPS (s-EPS) and bound-EPS (b-EPS). Previous studies found that EPS could form an important extracellular protective barrier on the algae cell surface, which can prevent toxic metals from invading the intracellular environment. Moreover, metals (e.g., Pb²⁺, Cd²⁺) also affect the production and composition of EPS. Additionally, EPS have a variety of functional groups (e.g., -COOH, -NH, -OH, etc.), which have significant effects on the adsorption of metal ions by EPS. However, the binding mechanisms of EPS with heavy metals in microalgae remain unknown. Here, we hypothesize that the EPS yield and components of Chlamydomonas reinhardtii increase significantly under the stress of heavy metals and this in turn contributes to higher metal sorption capacity. We conducted several experiments to verify our hypothesis. The tolerance of microalgae cells and the contents of EPS were evaluated under various metal treatments (Cd, Pb, Cu, Zn). To confirm the combination of EPS with heavy metals, Three-dimension excitation emission matrix fluorescence spectroscopy (3D-EEM) analyses were performed to characterize the components of EPS. Our data show that the total amount of EPS under the stress of Pb and Cu in the 4-d cultivation increased 193%, 46% respectively. When C. reinhardtii was inoculated in the TAP media with Cd²⁺, Zn²⁺, the content of proteins in EPS increased. The 3D-EEM analyses demonstrated that tyrosine-like substances and tryptophan were observed at the Ex/Em of 225/324 and 280/337 respectively, and the fluorescence intensity of EPS decreased gradually along with the increase of concentration of four metals. These results indicate that tyrosine-like substances and tryptophan in EPS may play an important role in the adsorption of heavy metals by microalgae. Overall, it can be concluded that the increase of EPS and their components in response of heavy metals may be one effective way to reduce the metals toxicity. Further research is onging to reveal the impact of EPS on the binding capcity of different heavy metals C. reinhardtii.