Mastering the change of grassland vegetation in Three-River Headwater Region has important guiding significance for grassland restoration and ecological construction project. This study analyzed the linear trend and inter-annual variation of GNDVI with altitudinal gradient in the Three-River Headwater (TRH) Region from 2000 to 2015, the purpose of which is to clarify the interannual fluctuation of alpine grassland in TRH Region and its responses to different hydrothermal combination. The GNDVI showed an increasing trend with large inter-annual variation (IAV) due to climate factors and biological processes of vegetation. The meadow determined the IAV of TRH GNDVI rather than the steppe, and the relative contribution of meadow and steppe to the IAV of TRH GNDVI was 75.4% and 24.6%, respectively. The annual mean value of GNDVI in TRH Region decreased with elevation, while the trend of GNDVI increased with elevation. Different elevations in the TRH region, meadow area and steppe area had differential relative contributions to the IAV of GNDVI, The relative contribution of GNDVI inter-annual variations in the low-altitudinal area was much greater than that in the high-altitudinal area. The IAV of GNDVI in low-altitude areas was dominated by precipitation, while that in high-altitude areas was dominated by temperature in the TRH region.