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• The rainfall-runoff relationship has changed under climate change and human activities in the TRH region. • The impacts climate indicators of rainfall-runoff ratio in the sub-basins of TRH region were different. • The contribution rates of human activity in the YRB were a little higher than that in the YARB. Identifying the impact of climate change and human activities on rainfall-runoff relationship in the Three-River Headwaters (TRH) region is essential for water source conservation, ecological security, and biodiversity maintenance of China and East Asia. In this paper, the runoff-rainfall ratio was used as the index of rainfall-runoff relationship. Different methods were employed to identify the impact indicators and quantitative assessment of rainfall-runoff relationship from 1957 to 2016. The results are the following: (1) the rainfall-runoff relationship has changed in the source regions of Yangtze River basin (YARB) and Yellow River (YRB), and runoff-rainfall ratio has increased by 2.9% and decreased by 3.2%, respectively; (2) the main climate indicators of runoff-rainfall ratio were rainfall, potential evaporation and sunshine hours in the YARB, and rainfall, potential evaporation and temperature in the YRB; (3) the contribution rates of human activity in the YRB were a little higher than that in the YARB, and the contribution rates of climate change and human activity were 90.9% and 9.1% in the YARB and 42.9% and 57.1% in the YRB. The results can provide invaluable information for managing water resources and taking ecological protection measures. [ABSTRACT FROM AUTHOR]