Supply and demand of ecosystem services can reflect the coordination level of regional eco-economic development. Spatially quantifying the supply and demand of ecosystem services and identifying the mismatch between them are significant to regional ecosystem sustainable management. Huangshui River Basin is an important part of ecological barrier construction in Northwest China. The balance between supply and demand of ecosystem services is directly related to the ecological security of Qinghai Province. Based on the comprehensive multi-source data, multiple biophysical models were used to quantitatively evaluate the supply and demand of food production, water yield, soil conservation and carbon sequestration in Huangshui River Basin. The aim was to show the spatial characteristics of the supply and demand of ecosystem services and to reveal the spatial aggregation pattern of supply-demand in the basin. The results show that:(1) The spatial distribution supply and demand of ecosystem service is highly heterogeneous in Huangshui basin. Except for water yield service,other ecosystem services can meet the internal demand of the basin, but there are still obvious deficit characteristics in some regions. (2) The supply-demand ratio of different ecosystem services is significantly different. It ranks from great to small as follows:carbon sequestration (0.118), food production (0.114), soil conservation (0.026), and water yield service (-0.021). There were significantly spatial differences in the supply-demand ratios of various services in the watershed. (3) The spatial mismatch between supply and demand was prominent in the Huangshui River Basin. Except for water yield service, other ecosystem services have "high supply and low demand" areas and "low supply and high demand" areas. (4) The type of land use affects the balance between supply and demand of all ecosystem services. To achieve the balance between supply and demand, the proportion of forest and grass land should be moderately increased and the expansion of cultivated land and urban land should be controlled. This paper systematically analyze the supply and demand of ecosystem services in Huangshui River Basin, which can provide theoretical support for ecosystem management and decision-making of land resource allocation.