Environmental footprint represents the degree of the impact of human development on the environment. Environmental footprint and human well-being are the important indicators to measure the sustainable development capacity of a region. This paper calculated the environmental footprint and environmental boundary of the Three-River Headwaters Region from 2000 to 2018, constructed a framework for evaluating human well-being in the Three-River Headwaters Region, and evaluated the coupling coordination relationship and the dynamic characteristics of the environmental footprint and human well-being in the Three-River Headwaters Region from 2000 to 2018. The quantitative relationship between the environmental footprint and human well-being in the Three-River Headwaters Region from 2000 to 2018 was obtained, which can provide scientific support for the relevant departments to formulate reasonable regional development strategies. The results indicated that: (1) Overall, the environmental footprint per capita of the Three River Headwaters Region from 2000 to 2018 exceeded the environmental boundary limit. (2) Human well-being in the Three River Headwaters Region indicated that social and cultural well-being was higher than safety, health, and basic needs in the order of importance. From 2000 to 2018, the overall human well-being in the Three-River Headwaters Region showed a fluctuating upward trend, of which safety and health fluctuated the most and contributed the most to the overall human well-being. (3) The coupling degree and coupling coordination degree between the environmental footprint and human well-being in the Three River Headwaters Region showed a fluctuating upward trend, and the level of correlation between the two gradually increased, and gradually tended to be coordinated.