National parks have implemented restrictive measures on human activities to protect the ecological environment, which has changed the livelihood dependence and strategies of farmers and herders. Exploring the impact of livelihood strategies on the human well-being of farmers and herdsmen within the national park construction area can help to better develop livelihood intervention measures to achieve sustainable livelihoods for farmers and herders. A total of 329 farmers and herders in Qilian Mountain National Park in China were investigated, and one-way ANOVA and ordinary least squares were employed to analyze the impact of farmers’ and herders’ livelihood strategies on human well-being in different periods of national park construction. Our results show that the livelihood strategies of farmers and herders play an important role in their well-being. Farmers and herders adopted diversified livelihood strategies after the National Park System Pilot officially launched (after 2015). The well-being advantage of adopting a diversified livelihood strategy became evident over time. Specifically, farmers and herders adopting the settlement diversification livelihood strategy were able to better adapt to the development concept of the national park after the national park pilot was officially established (after 2017). However, nomadic, settled agricultural, and pastoral households were always at a disadvantage in terms of well-being. In addition, distance, nationality, gender, and education level were important factors affecting the well-being of farmers and herders. The results of this study are helpful for improving our understanding of the influence of livelihood strategies on the well-being of farmers and herders and the related challenges they face in the construction of national parks.