In order to obtain the optimal conditions for ammonia nitrogen (AN) wastewater treatment by bio filter (BF), the effects of C/N, pH, and hydraulic load (HL) on the AN degradation were studied by Response Surface Methodology (RSM). Central Composite Design (CCD) experiments were conducted, and the response of the AN removal rates were fitted to a second-order polynomial model. The analysis of variance showed that the model was accurate and reliable. Through model fitting, the optimal condition for AN removal was: C/N of 18.95, pH of 7.78, and HL of 1.04 d-1. The maximum AN removal rate predicted by the model was 91.90%, accorded with the experimental verification value of 91.37% under the optimal condition. The research provided valuable demonstration for optimizing process parameters on AN removal in BF.