[Objective] To study the ammonia nitrogen (NH4+-N) removal characteristic, extraction and activity of its key enzymes in Acinetobacter sp. Y1. [Methods] Sodium citrate and ammonium sulfate were used as the sole carbon and nitrogen sources respectively to investigate NH4+-N removal characteristic of strain Y1. The ultrasonic conditions were optimized using orthogonal experiment, and the crude enzyme obtained from ultrasonic crushing method and osmotic shock method were compared by SDS-PAGE, then the activity of key enzymes——hydroxylamine oxidoreductase (HAO), nitrite reductase (NIR) and nitrate reductase (NAR) were measured. [Results] Bacteria density (OD600) was 1.280, and NH4+-N, total nitrogen (TN), COD removal rate can reach up to 98%, 94% and 92% respectively of strain Y1 in 24 h. Hydroxylamine, nitrite nitrogen and nitrate nitrogen in nitrification process were detected without accumulation, and N2 was produced in the process of denitrification. The optimal ultrasonic conditions were: ultrasonic power 50 W, work/interval time 4/7 s, OD600=1.250 and total working time 20 min, and the corresponding specific activities of HAO, NIR and NAR were 0.011, 0.002, 0.018 U/mg, respectively. Specific activity of HAO obtained by osmotic shock method was 0.067 U/mg. [Conclusion] NH4+-N, TN and COD were removed simultaneously with high efficiency by strain Y1. HAO, NIR and NAR activities were detected after optimizing the ultrasonic conditions. And it was better to use osmotic shock method for HAO extraction than ultrasonic crushing method.