Based on the measured and related data from meteorological and hydrological stations in the Three-River Headwaters Region during 1956—2012, the variation characteristics of precipitation, temperature, NDVI (normal difference vegetation index) and runoff were analyzed by using the linear regression method, Mann-Kendall test method and Spearman test method. Moreover, the influences of climate and the underlying surface to runoff were analyzed by using the weighting factor method based on Budyko hypothesis. The results indicated that the annual precipitation showed a significant increase trend in the source region of the Yangtze River and Lancang River, and the annual mean temperature showed a significant warming trend in the Three-River Headwaters Region. The annual NDVI showed a significant increase trend in the source region of the Yellow River. In addition, the changing slopes of annual runoff in the source region of the Yangtze River, Yellow River and Lancang River were 6.54×108m3,—2.05×108m3 and 1.54 ×108m3 per 10 years, respectively, but these trends were not significant. The weighting factor method used in this study can not only accurately estimate the annual mean runoff, but also need not to assume that the climate and underlying surface changes have the same contributions to runoff changes. Overall, both climate and underlying surface showed a positive contribution to runoff changes in the source region of Yangtze River and Yellow River. However, in the source region of Lancang River, the underlying surface is positive contributing factor to runoff variations when the weighting factor (i.e., ) was 1.0, whereas it was a negative contributing factor when was 0.5 and 0. The contributions of climate change on runoff in the source region of the Yangtze River, Yellow River and Lancang River were 55.21%~92.85%, 52.47%~68.50%, and 89.91%~124.58%, respectively. Meanwhile, our results emphasized that the changes of runoff in the Three-River Headwaters Region were mainly affected by climate change. Precipitation is the main controlling factor of climate that influences runoff, and vegetation cover is the main controlling factor of underlying surface that influences runoff.