To explore the effects of organic fertilizer nitrogen replacing chemical fertilizer nitrogen on the growth, carbon and nitrogen metabolism of hulless barley in the Qinghai Tibet Plateau, pot experiments were conducted under the same nitrogen, phosphorus, and potassium application rates. The differences in growth characteristics, leaf carbon and nitrogen balance, and nitrogen metabolism enzyme activity of hulless barley variety Kunlun 14 were compared and analyzed among different proportions of organic fertilizer nitrogen replacing chemical fertilizer nitrogen (0%, 40%, and 100%, designated as ORF0, ORF40, and ORF100) treatments. The results showed that compared with ORF0, the root length and old leaf carbon to nitrogen ratio of hulless barley significantly increased under ORF40 treatment, while the changes in plant height, aboveground or root biomass were not significant. Under ORF100 treatment, plant height and aboveground biomass significantly decreased, but root length and biomass significantly increased (P<0.05), and carbon to nitrogen ratio of new leaves significantly increased. Compared with ORF0 treatment , the activities of glutamate synthase (GOGAT) and glutamate dehydrogenase (GDH) in old leaves under ORF40 treatment were increased by 1.18%-2.53% and 17.39%-39.40%, respectively, from seedling stage to flowering stage. The GOGAT activity of new and old leaves under ORF100 treatment showed no significant difference, while the GDH activity significantly decreased. The activity of glutamine synthetase (GS) in the new leaves under ORF40 treatment was higher than that under ORF0 treatment during the heading and flowering stages. The activity of glutamine aminotransferase (GOT) in the new leaves was significantly increased by 22.22%-35.29% from seedling stage to flowering stage (P<0.05). The GS activity of both new and old leaves under ORF100 treatment significantly decreased. Overall, compared with the ORF0 treatment, the ORF40 treatment showed less variation in root length and plant height of hulless barley. The leaves showed more stable C/N, and the activities of GS and GDH increased in the old leaves, while the activities of GS and GOT increased in the new leaves. Both the new and old leaves under ORF40 treatment maintained a certain level of nitrogen metabolism ability, with the nitrogen metabolism ability of the old leaves being more stable, indicating that an appropriate proportion of organic fertilizer replacing chemical fertilizers is helpful for the stable growth and carbon and nitrogen metabolism of hulless barley plants.