Abstract: Ammonia volatilization from highland barley fields can contaminate the atmosphere and lead to the reduction of nitrogen use efficiency. Field trials were conducted to estimate the influences of different levels of nitrogen application on ammonia volatilization flux, NH₃ volatilization loss, nitrogen efficiency and grain yield in highland barley farmland ecosystem in Tibet. Results show that the yield of highland barley is significantly and positively correlated with nitrogen application (P < 0.05). The contribution rate of nitrogen fertilizer was 32.13%, and the agronomic benefit of nitrogen fertilizer was 3.62-4.78 kg· kg^-1; The total accumulation of ammonia volatilization was 3.38-21.51 kg·hm^-2, The nitrogen loss rate caused by ammonia volatilization was 10.75%-15.49%, which increased significantly with the increase of nitrogen application (P < 0.01); Ammonia volatilization mainly occurred in the basal fertilizer period, the ammonia volatilization stage of basal fertilizer was 44 days, and the volatilization peak appeared successively on the 10th day, and the ammonia volatilization of topdressing fertilizer was concentrated with 7days after fertilization. Comprehensively considering the production benefits and environmental safety based on ammonia volatilization, it is suggested that the application amount of pure N in this region is 40.02 kg·hm^-2 for base fertilizer, 13.32-26.70 kg·hm^-2 for top-dressing during knotting, 40.02 kg·hm^-2 for top-dressing during heading grain.