A denitrifying ammonium oxidation (DEAMOX) process has been regarded as an innovative process to simultaneously treat ammonia and nitrate containing wastewaters, whereas very limited research has focused on its application in biofilm system. In this research, a novel DEAMOX process was established with fixed sponge carriers in a sequencing biofilm batch reactor (SBBR). To investigate biofilm formation process and characteristics can encourage further research on DEAMOX system optimization, deteriorated performance recovery strategies and application with actual wastewater. Total nitrogen removal efficiency was maintained at 93.0 % after 240 days of operation. With biofilm growth, the protein-like extracellular polymeric substances (EPS) and tightly-bound EPS (TB-EPS) of biofilms increased from 65.6 to 46.1, to 179.6 and 142.0 mg gVSS(-1), respectively, revealing that protein-like substances and TB-EPS promote biofilm formation. The mechanism of biofilm formation was discussed by analyzing the morphological development and functional bacterial activities of biofilms. Furthermore, high anammox activity was obtained in biofilms with specific NH4+-N removal rates over 4.29 mgN gVSS(-1)h(-1), which were significantly higher than in suspended sludge (2.56 mgN gvss(-1)h(-1)). Quantitative polymerase chain reaction results showed that the abundance of anammox bacteria in biofilms increased from 1.87 % to 11.48 % with biofilm growth. These results imply that mature biofilms formed on carriers and the anammox bacteria were sufficient enriched in DEAMOX-SBBR system. (C) 2018, The Society for Biotechnology, Japan. All rights reserved.