Abstract: To explore the effects of different nitrogen application rates on soil organic carbon and enzyme activities in Lhasa farmland, a field experiment was conducted with five treatments (T1-T5): 0, 75, 150, 225 kg N /hm2, and 135 kg P2O5/hm2, respectively. The application rates of 225 kg N/hm2 and 135 kg P2O5/hm2 were considered as the suitable fertilization amounts. 0-20 cm soil samples were collected before and after harvest of highland barley, to determine the biochemical properties including total organic carbon, labile organic carbon, humus carbon and humic acid content, and sucrase, polyphenol oxidase and urease activities, respectively. The results showed that the total amount of soil organic carbon and the humification rate were low in Lhasa farmland, however, the proportion of active organic carbon was large. Short- term application of nitrogen fertilizer increased soil total organic carbon by raising soil active organic carbon and improved soil HA/FA, however, reduced soil humus carbon content. In the short term, the application of nitrogen fertilizer might increase the soil organic carbon content in local farmland, T4 can optimize the soil microenvironment, and improve soil enzyme activities and soil organic carbon accumulation.