Worldwide, anthropogenic modification of rivers and their flow imperils aquatic ecosystem integrity. The River Murray in south-eastern Australia flows from the Australian Alps to the Southern Ocean, traversing >2000 km of temperate and semi-arid climatic regions. The Murray is one of the world’s most regulated rivers, having been extensively modified for navigation and consumptive water use. Headwater dams, weirs, water abstraction and tidal barrages have transformed the river’s hydrology and hydraulic nature, such that: 1) the mid–upper river (~1000 km) is subject to seasonal flow inversion, 2) the lower river is an 800 km reach of sequential weir pools, and 3) end-of-system discharge is ~30% of natural. Together, these factors have contributed to long-term decline in the health of the river’s aquatic ecosystems. Over the past two decades, cross-jurisdictional river management has included using environmental water allocations aimed at rehabilitating ecosystem health. To be effective, this management requires a sound understanding of flow-ecology relationships. Despite numerous studies examining ecological responses to flow at specific locations across the River Murray, a consistent, integrated assessment along the entire river has been lacking. In 2021, the River Murray Channel Monitoring Project was established to investigate the influence of flow on riverine ecosystem structure and function, from annual–decadal timescales, along four hydro-geomorphologically distinct regions of the River Murray. A range of physico-chemical and biological parameters are measured, including nutrients, ecosystem metabolism, phytoplankton and zooplankton production, and fish population demographics. A key objective of the program is to understand how flow may influence ecosystem function across trophic levels; for example, how primary and secondary productivity relate to fish recruitment. Here we present: the architecture of the program, preliminary results after 3-years initial monitoring, prospects for multi-year analysis, and the utility of our findings for flow management in the River Murray and elsewhere.