Ecosystem service flows link ecosystems to human well-being, with freshwater being a key provisioning service. Focusing on the Qinghai–Tibet Plateau as Asian Water Tower, this study examines pathways of water supply service flow under dynamic supply–demand dynamics and their implications for ecological management. Current research often oversimplifies spatial processes, lacks multi-scale analysis, and overlooks factors such as beneficiary competition and opportunity costs in payment schemes. To address these gaps, we (a) evaluated freshwater supply, demand, and their relationships; (b) modeled service flows using an improved resistance surface and algorithms (D8-Dijkstra-MCR and 2SFCA) across multiple scales; and (c) refined payment for ecosystem services (PES) by integrating Jensen–Shannon divergence with opportunity costs and socioeconomic factors. Main findings reveal the following: (a) At finer spatial scales, the heterogeneity of supply and demand intensifies, generally following a gradient from high in the southeast to low in the northwest. (b) Service flow networks grow more complex with increasing resolution. (c) Flow volumes exhibit scale-dependent variation, highest in northwestern counties, concentrated around city clusters at the municipal scale, and spatially clustered in southeastern basins at the watershed level. (d) The service flow-based PES and the current watershed PES exhibit convergence in regional payment structures. This study underscores the need for multi-scale management and differentiated PES schemes to support regional sustainability.