Detecting the effects of climate changes and human activities on river regimes would help to identify the original driving forces of hydrological disturbances and highlight the mechanisms of such hydrological processes under changing conditions. Two non-parametric tests (Mann-Kendall and Pettitt) were applied to detect the change point of runoff (1964–2006) in the upper and middle reaches of the Heihe River basin. The change point was determined to be 1980, which divided the period into two parts (the baseline period and the change period). Double mass curve and hydrological sensitivity-based methods were used to separate the impacts of climate changes and human activities on runoff variation. The results demonstrated that human activities were the dominant force affecting runoff variation in the upper and middle reaches of the basin. At the sub-basin level, climate changes played a more significant role in the upstream region, while human activities dominated in the midstream region. Therefore, different countermeasures should be taken in the upstream and midstream regions to ensure sustainable water resource development in the Heihe River basin.