The upper reaches of the Yellow River serves as a crucial ecological barrier and functional zone in China. Investigating the temporal and spatial changes of the ecosystem services, as well as the balanced and coordinated relationships among them, has significant importance in achieving ecological protection and promoting high-quality development in the Yellow River basin. This article quantitatively analyzed the spatio-temporal distribution characteristics and the trade-offs and synergies of the ecosystem services in the upper reaches of the Yellow River basin from 2000 to 2020 based on data including land use, soil, meteorology and others, with InVESTmodel, correlation analysis, and spatial autocorrelation analysis. The results displayed as follow: (1) The overall performance of various ecosystem services in the upper reaches of the Yellow River basin exhibited a distribution pattern of "higher in the southwest and lower in the northeast". The high-value areas were predominantly located in the southwest of the basin, while the low-value areas were primarily distributed in the northeast of the basin. (2) During the study period, various ecosystem services in the upper reaches of the Yellow River exhibited fluctuating trends. Water yield and soil conservation showed an overall decreasing tendency, while habitat quality and carbon storage demonstrated an increasing trend. (3) There were varying degrees of correlation among different ecosystem services. Water yield exhibited a trade-off relationship with habitat quality and carbon storage, indicating a dynamic balance between these factors. Additionally, there was a synergistic relationship between water yield and soil conservation, habitat quality and carbon storage, as well as soil conservation and carbon storage. (4) In the spatial distribution pattern, the trade-offs and synergies among various ecosystem services exhibited significantly spatial heterogeneity and scale effects, with substantial differences among different zones. The study can provide guidance for scientifically managing regional ecosystems and promoting sustainable development within watersheds.