In this study, the competitive mechanism of ammonia, iron and manganese for dissolved oxygen (DO) in a biofilter was investigated, and a new start-up method of a biofilter for ammonia, iron and manganese removal was approved, which can effectively shorten the start-up period from 3–4 months to 51 days. The results demonstrated that when DO was sufficient (about 8 mg · L−1), ammonia, iron and manganese could be completely removed. When DO decreased from 6.5 to 4 mg · L−1, the concentration of ammonia in the effluent increased accordingly, though iron and manganese were removed efficiently. When DO was as low as 3 mg · L−1, only iron was removed, whereas most of the ammonia and manganese still existed in the effluent. In addition, the oxidizing rates of the pollutants were not affected significantly with DO decrease. Turbidity removal in the biofilter was also investigated, and the results demonstrated that the turbidity decreased to less than 0.5 NTU at 0.4 m depth of the filter.