Using 13C pulsed labeling technique, we examined the biomass and carbon accumulation of different organs as well as the distribution characteristics of 13C assimilate of 6-year-old Korla fragrant pear trees under three nitrogen application levels, i.e., 150, 300, and 450 kg N·hm-2 (marked as N1, N2, and N3, respectively). Results showed that the biomass, carbon accumulation, 13C fixation and leaf assimilation capacity of the whole pear tree increased while root to shoot ratio decreased with increasing nitrogen application. Both biomass and carbon accumulation amount of reproductive organs (i.e., fruits) were the highest under N2 treatment. The 13C content and distribution rate of each organ changed dynamically along with increasing nitrogen application. At the new shoot growing stage, leaves and roots had stronger competitive abilities for photosynthate, with 13C distribution rates being the highest under N1 treatment. During fruit swelling and mature stages, leaves and fruits were more competitive, with 13C content and distribution rate in leaves being the highest under N3 treatment and those in fruits being the highest under N2 treatment. According to the absorption and distribution characteristics of carbon assimilate across organs under the three nitrogen application levels, the optimal nitrogen application level for achieving high fruit yield in the 6-year-old Korla fragrant pear tree orchard is recommended as 300 kg·hm-2 .