Abstract: In conventional urban sewage treatment plants, nitrate nitrogen and a wide range of organic micropollutants (OMPs), including endocrine disruptors and pharmaceuticals, frequently coexist in the effluent from secondary treatment processes. These compounds have been demonstrated to exert significant effects on various microorganisms. However, their influence on the advanced denitrification process remains to be comprehensively elucidated. This study aimed to investigate the response of the denitrification process to OMPs through the construction and operation of three denitrification moving bed biofilm reactors. The reactors were employed to evaluate the denitrification removal rate, assess the efficiency of micropollutant removal, analyze the response of extracellular polymeric substances (EPS) in the biofilm, and examine variations in key enzyme activities within the electron transport chain. The findings revealed that the presence of OMPs had a detrimental impact on denitrification efficiency, resulting in instability and a significant reduction in carbon and nitrogen removal rates to below 70%. Concurrently, the removal efficiencies of micropollutants such as ethinyl estradiol (EE), estriol (E3), and diclofenac (DCF) were found to exceed 75%, whereas the removal rates of carbamazepine (CBZ) varied between 20% and 44%. This suggests that different OMPs are handled differently by the denitrification system. During the introduction of micropollutants into the systems, it was observed that microorganisms initially utilized a portion of the EPS as a nutrient source, indicating an important role of EPS in microbial adaptation. Subsequently, in response to the stress induced by OMPs, these microorganisms secreted additional EPS. This response underscores that EPS plays a crucial role in the microbial adaptation to micropollutant stress. Moreover, the activity of key enzymes within the electron transport chain was inhibited due to the presence of micropollutants, thus reducing electron transport efficiency and consequently impairing the overall denitrification performance. The study further demonstrated that elevated concentrations of organic micropollutants exerted a more pronounced negative impact on denitrification efficiency, EPS content, and key enzyme activities within the electron transport chain, leading to an accelerated metabolic response from microorganisms. This study provides valuable insights into the complex interactions between micropollutants and denitrification processes, particularly regarding the effect of OMPs on denitrification. It highlights an urgent need for enhanced strategies aimed at improving nitrogen removal alongside co-removal of these contaminants during wastewater treatment. Understanding these impacts is essential for developing more effective treatment technologies to address challenges posed by both nitrate and OMPs present in wastewater effluents.