The vertical distributions of nitrifying bacteria in aerobic fixed biofilm were investigated to evaluate the relationship between nitrification performance and microbial community at different HRT. Fluorescent in situ hybridization (FISH) and portable ion selective microelectrode system were adopted to analyze microbial communities and ions profiles according to the biofilm depth. Cilia media packed MLE (Modified Ludzack-Ettinger) like reactor composed of anoxic, aerobic Ⅰ/Ⅱ was operated with synthetic wastewater having COD 200 mg/L and NH4 +-N 40 mg/L at HRT of 6 hrs and 4 hrs. Total biofilm thickness of aerobic Ⅰ, Ⅱ reactor at 4 hrs condition was over two times than that of 6 hrs condition due to the sufficient substrate supply at 4 hrs condition (6 hrs; aerobic Ⅰ 380 ㎛ and Ⅱ 400 ㎛, 4 hrs; aerobic Ⅰ 830 ㎛ and Ⅱ 1040 ㎛). As deepen the biofilm detection point, the ratio of ammonia oxidizing bacteria (AOB) was decreased while the ratio of nitrite oxidizing bacteria (NOB) was maintained similar distribution at both HRT condition. The ratio of AOB was higher at 4 hrs than 6 hrs condition and NH4 +-N removal efficiency was also higher at 4 hrs with 89.2% than 65.4% of 6 hrs. However, the ratio of NOB was decreased when HRT was reduced from 6 hrs to 4 hrs and NO2 --N accumulation was observed at 4 hrs condition. Therefore, it is considered that insufficient HRT condition could supply sufficient substrate and enrichment of AOB in all depth of fixed biofilm but cause decrease of NOB and nitrite accumulation.