To achieve an appropriate mixture of ammonium and nitrite for anaerobic ammonium oxidation (ANAMMOX), 50% partial nitritation was optimized in a fixed bed biofilm reactor treating synthetic wastewater. Results suggested that 50% partial nitritation could be achieved by stepwise increases of influent NH 4 + -N at pH of 7.8±0.2, temperature of 30±1°C and dissolved oxygen (DO) of 0.5–0.8mgl −1 . Hydraulic retention time (HRT) and influent alkalinity did significantly affect partial nitritation. At HRT 12h, 50% partial nitritation could be kept stable, regardless of influent NH 4 + -N variation, by controlling the influent HCO 3 − /NH 4 + molar ratio at 1:1. The fluorescent in situ hybridization (FISH) results indicated the abundance of evolution of ammonia-oxidizing bacteria (AOB) and the nitrite-oxidizing bacteria (NOB) coincided well with the performance of partial nitritation. Furthermore, the AOB were highly affiliated with Nitrosomonas spp. and Nitrosospira spp. dominated (64.1%) in the biofilm with a compact structure during the stable 50% partial nitritation period.