The contribution of biodiversity to human well-being is key to exploring the relationships between biodiversity, ecosystem services (ES), and human well-being. In this work, a composite index, termed the human well-being index (HWI), was constructed for evaluating the contribution of biodiversity to human well-being in the Sanjiangyuan area. This index consists of material, ecological regulation, and spiritual and cultural contributions, represented by the material index (MI), the ecological regulation index (ERI), and the spiritual and cultural index (SCI), respectively. The system was further used to evaluate the spatiotemporal distribution of human well-being at the county level in 2000, 2010, and 2020. HWI increased steadily across Sanjiangyuan over the study period, especially in the western and northeastern counties; its center of gravity shifted in the northward direction. The MI increased (decreased) in the west and northeast (southeast); its center of gravity shifted in the northeast direction. All counties showing changes in the ERI were located in the eastern part of Sanjiangyuan. The center of gravity of ERI did not change significantly. The SCI increased steadily across the study area, but was high in the west and low in the east; the center of gravity shifted in the northwest direction. The study findings can contribute toward quantifying biodiversity contributions to human well-being and the formulation of biodiversity conservation policies.