To investigate the influences of free ammonia on nitrogen removal, the contents of extracellular polymeric substances and the chemical composition (proteins, polysaccharides, and DNA), four laboratory-scale sequencing batch reactors fed with synthetic wastewater were operated at free ammonia concentrations of 0.5, 5, 10 and 15 mg/L (R_0.5, R₅, R₁₀ and R₁₅, respectively). Results showed that high nitrogen removal efficiencies (97.6%~99.4%) were achieved under four free ammonia concentrations. Free ammonia had a significant impact on the three kinds of extracellular polymeric substance (loosely bound extracellular polymeric substance, tightly bound extracellular polymeric substance and total extracellular polymeric substance, respectively) and their composition. Results showed that with the increased of initial free ammonia concentrations from 0.5 to 10 mg/L, the production of three kinds of extracellular polymeric substance and their components were significantly increased. Further increased of free ammonia concentrations to 15 mg/L caused a decreased trend of them. Moreover, proteins were the main component of loosely bound extracellular polymeric substance, while polysaccharides dominated in tightly bound extracellular polymeric substance and total extracellular polymeric substance under different free ammonia concentrations. It was found that the contents of three kinds of extracellular polymeric substance and their components showed a similar variation tendency to the NO_x^-—N contents during the whole cycle under four free ammonia concentrations.