The aim of the present work was to evaluate the effects of carbon sources and chemical oxygen demand (COD)/NO2--N ratios on the anammox-denitrification coupling process of the simultaneous partial nitrification, anammox and denitrification (SNAD) biofilm. Also, the anammox activities of the SNAD biofilm were investigated under different temperature. Kaldnes rings taken from the SNAD biofilm reactor were operated in batch tests to determine the nitrogen removal rates. As a result, with the carbon source of sodium acetate, the appropriate COD/NO2--N ratios for the anammox-denitrification coupling process were 1 and 2. With the COD/NO2--N ratios of 1, 2, 3, 4 and 5, the corresponding NO2--N consumption via anammox was 87.1%, 52.2%, 29.3%, 23.7% and 16.3%, respectively. However, with the carbon source of sodium propionate and glucose, the anammox bacteria was found to perform higher nitrite competitive ability than denitrifiers at the COD/NO2--N ratio of 5. Also, the SNAD biofilm could perform anammox activity at 15 °C with the nitrogen removal rate of 0.071 kg total inorganic nitrogen per kg volatile suspended solids per day. These results indicated that the SNAD biofilm process might be feasible for the treatment of municipal wastewater at normal temperature.