Ecosystem provides humans with goods and services that form the foundation of sustainable human development. The study of ecosystem services supply, demand and consumption as well as flow characteristics and relationships, is significant for regional ecosystem management and the development of ecological compensation policies. This study adopts the Yellow River Basin as a case study, calculates ecosystem service supply and demand indices for 2021, and identifies spatial flow paths and quantities of usage from the supply area to the demand area using various methods and models, including the Minimum Cumulative Resistance (MCR) model, the gravity model, and the urban breakpoint theory. The results indicated that: 1). The ecosystem service supply per unit area in the upstream region of the Yellow River exceeds that of the middle and lower reaches. Meanwhile, the regional demand for ecosystem services in the lower stream of the Yellow River has surpassed that in the middle and upper stream, demonstrating spatial heterogeneity in both supply and demand across the Yellow River Basin. 2). Additionally, in terms of the balance between supply and demand of ecosystem services in the Yellow River, the upstream area primarily exhibits a surplus of ecosystem service in supply. Most of the cities in Shaanxi Province, Shanxi Province, and Ningxia Hui Autonomous Region in the middle stream maintain a balance in ecosystem service supply, while Henan Province and Shandong Province in the downstream experiencing a deficit in ecosystem service supply. 3). In 2021, the total value of ecosystem service flows in the Yellow River Basin was 579.43 billion yuan. This total includes 222.39 billion yuan from Gansu Province and 165.03 billion yuan from Qinghai Province, with ecological services predominantly transferred to Shanxi Province. This study provides theoretical support for the construction of a horizontal ecological compensation mechanism in the Yellow River Basin, and suggests optimizing the transfer payments for key ecological functional areas in Qinghai and Gansu Provinces, so as to form a benign situation in which the beneficiaries pay and the protectors are reasonably compensated. 4). The intensity of ecosystem service flows demonstrates distance-decay characteristics, with long-distance transmission from supply to demand areas remaining constrained. Ecological restoration around ecosystem service demand areas should be enhanced to improve human ecological well-being in the demand areas by increasing the supply of ecosystem services in the vicinity.