The start-up and operation of single-stage autotrophic nitrogen removal process in upflow double layer filler reactor were successfully established in this study after 139 days. Zeolite and polyurethane foam were used as reactor filler. Synthetic high ammonia nitrogen (110~130mg/L) wastewater was fed to the reactor. Ratio of filler zeolite and polyurethane foam height in No.1and No.2 reactor was 2:3 and 3:2, resprctively. The results showed that the total nitrogen removal efficiency of No.1reactor reached 84.4%, while in the No.2 reactor 81.8%, with a total nitrogen removal load achieved 0.15 and 0.14kgN/(m³·d), respectively. Without adding organic matter into influent, compared with No. 1reactor, △NO₃^--N/△NH₄⁺-N of No.2 reactor has been stable around 0.11. The total nitrogen removal efficiency of two reactors both increased with the addition of organic carbon source. It was indicated that appropriate concentration of COD can enhance autotrophic nitrogen removal performance. After backwashing, the NO₃^--N concentration of No. 1 reactor decreased from 17.61mg/L to below 10mg/L. It was showed that backwashing can effectively wash out NOB, and the combination of backwashing and other NOB suppression strategies can better maintain the long-term stable operation of single-stage autotrophic nitrogen removal process.