Sulfonamides (SAs) are common antimicrobial drugs, which are frequently detected in surface water systems, and are difficult to degrade, posing a potential threat to the aquatic environment. However, little is known about the potential adverse effects of SAs on non-target organisms (e.g., microalgae) in the aquatic ecosystem. In this study, the effect of SAs (sulfadiazine (SD), sulfamerazine (SM1), and sulfamethazine (SM2) at 1, 5, 20, and 50 mg/L concentrations, respectively) on the freshwater microalga Dictyosphaerium sp. was investigated, with respect to changes of biomass and chlorophyll a content and induction of extracellular polymer substances (EPS), including protein and polysaccharide contents. At the same time, the residue of SAs was determined. The results showed that Dictyosphaerium sp. was tolerant to the three SAs, and the chlorophyll a content in Dictyosphaerium sp. significantly decreased on day 7, followed by a “compensation phenomena”. The increase in protein and polysaccharide contents played a defensive role in Dictyosphaerium sp. against antibiotic stress, and there was a strong positive correlation between polysaccharide contents and antibiotic concentrations. Dictyosphaerium sp. exhibited 35%–45%, 30%–42%, and 26%–51% removal of SD, SM1, and SM2, respectively. This study is helpful to understand the changes of EPS in the defense process of microalgae under the action of antibiotics, and provides a new insight for the ecological removal of antibiotic pollution in natural surface water system.