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In view of the challenges faced by the ecological security of cultivated land on the Qinghai-Tibet Plateau, an evaluation model for the ecological security of cultivated land in “one river and two tributaries” in Tibet was established to clarify the impact of agricultural development on the ecological security of cultivated land. The ecological security of cultivated land index system was proposed based on pressure-state-response (PSR) model, consisting of three elements and 13 indicators. The spatial and temporal change pattern of cultivated land ecological security from 1985 to 2013 was analyzed, and the obstacle factors of cultivated land ecological security in different regions were probed. Results showed that the intensity of agricultural development in the “one river and two tributaries” region of Tibet was relatively high, especially since 2000, when the application of chemical fertilizers and pesticides brought huge hidden dangers to ecological security. Before the 21th century, cultivated land ecological security in all counties was basically at the “excellent” level. From 2000 to 2010, the level of cultivated land ecological security in all counties was decreased and gradually changed to “poor”. Then, cultivated land ecological security was improved after 2010 and gradually recovered to “excellent” or “good”. The grade of cultivated land ecological security in the “one river and two tributaries” areas as a whole was relatively consistent, but the level of ecological security in a few counties was relatively backward. The obstacle factors of regional cultivated land ecological security both had a certain regional similarity and an obvious spatial heterogeneity. The application of nitrogen and phosphorus fertilizer was the main obstacle to the ecological security of cultivated land. The research result can reveal the historical and realistic state of the evolution of cultivated land ecological security in the plateau region and provide a basis for formulating differentiated ecological security regulation strategies.