The three rivers source region, known as the “Water Tower of China,” plays a crucial role in ensuring regional ecological security through its dynamic ecological barrier functions. This study quantitatively evaluated the evolution of four core functions—carbon sequestration, water conservation, soil and water conservation, and biodiversity—using land use data from 2000 and 2018, combined with multi-source geographic information and meteorological data, through the InVEST model and principal component analysis. The results showed that: (1) Total carbon storage increased significantly from 753.7 Tg in 2000 to 781.2 Tg in 2018, with an increment of 27.5 Tg. Forest land (49.689%), grassland (27.709%), and unused land (18.653%) constitute the core carbon reservoirs, maintaining a spatial pattern of “higher in the southeast and lower in the northwest.” (2) The capacity for water conservation showed remarkable improvement, with average runoff increasing from 137 to 229 mm (a 67.2% growth). The spatial distribution pattern evolved from fragmented to concentric circles radiating outward from central areas (Nangqian County and Yushu City). (3) Soil and water conservation exhibited significant regional heterogeneity: Although erosion was reduced in local areas like the Tongtian River Basin, the total bare soil erosion increased from 1,434,479.405 million tons to 1,650,740.271 million tons, while total soil erosion rose from 215,771.375 million tons to 295,158.724 million tons, indicating intensified erosion pressure in human-affected southeastern areas. (4) There was a steady improvement in biodiversity maintenance: The average habitat quality index rose from 0.891 to 0.907, while the average relative habitat degradation level decreased from 0.008 to 0.007. However, degradation hotspots around roads and urban areas persist. The comprehensive ecological barrier function index indicates that ecosystem services in the three rivers source region showed positive trends between 2000 and 2018, demonstrating the effectiveness of ecological engineering initiatives and providing data support for precision ecological management.