Chlortetracycline and oxytetracycline have high detection rates in wastewater treatment plants, usually coexisting in wastewater. Microbial extracellular polymeric substances (EPS) are the protective layers of the microbial cells against harmful substances, which play an important role in microbial life activities. Chlortetracycline and oxytetracycline may have a certain impact on microbial EPS. Nevertheless, there are few studies on the effects of chlortetracycline and oxytetracycline on microbial EPS during biological phosphorus removal. Three mixtures with different rates (L1, L2 and L3) were designed by direct equalization ray method to investigate the effects of chlortetracycline and oxytetracycline on microbial EPS. The effects of chlortetracycline and oxytetracycline and their mixtures on proteins and polysaccharides of EPS were studied. Three-dimensional fluorescence spectroscopy (3D-EEM) and Fourier transform infrared spectroscopy (FT-IR) was used to analyze the effects of chlortetracycline and oxytetracycline on the composition and structure of EPS. The results showed that, with the increase in concentration and the prolongation of reaction time, the performance of the biological phosphorus removal system gradually deteriorated. The contents of protein and polysaccharide of EPS increased first and then decreased, and the protein content was higher than that of polysaccharide. 3D-EEM analysis showed that, with the increase of chlortetracycline, oxytetracycline and their mixtures, the fluorescence intensity of protein-like substances of EPS first increased and then decreased, and humic and fulvic acid-like substances were produced when the concentration was higher. There was antagonism between chlortetracycline and oxytetracycline, so the fluorescence intensity of the mixture was weaker than that of the single action, and only the ratio L3 did not appear as a humic acid and fulvic acid substance. FTIR analysis showed that the CO stretching vibration of 1 653 cm-1 amido Ⅰ band,the CO symmetric stretching of 1 403 cm-1 amido Ⅱ band,the PO stretching vibration of 1 266 cm-1 and the carbohydrate C—OH stretching vibration of 1 100 cm-1 in EPS were affected by chlortetracycline,oxytetracycline and their mixtures. The antagonism between chlortetracycline and oxytetracycline causes differences in functional groups. The P—OH stretching vibration of 1004 cm-1 only exists in oxytetracycline,ratio L2 and ratio L3,The P—O stretching vibration of 900 cm-1 only exists in chlortetracycline,oxytetracycline,ratio L2 and ratio L3. It was absent for 770 cm-1 amino acid COO variable angle vibration only in chlortetracycline. This study will provide a theoretical basis for the scientific evaluation of the mechanism of effects of chlortetracycline and oxytetracycline and their mixed pollutants on microbial EPS during biological phosphorus removal.