The files provided here are the supporting data for analyses presented in “Comprehensive Environmental Flows Assessment for Multi-guilds in the Riparian Habitats of the Yangtze River” a manuscript submitted for publication in the journal of Water Resources Research. The updated data could be divided into five categories: 1) Hydraulic model data in the excel version named “Hydraulic model data” for setup, calibration and verification for the Hydraulic model, which includes bathymetry data in the sheet named “bathymetry data” for establishment of the digital bathymetry using MIKE21 module in DHIMIKE software in the Jianli reach of Yangtze River, and the depth and velocity surveyed data on April 11 and 12, 2019 in the sheet named “depth and velocity” used to calibrate and verify the hydraulic model, and the calibrated Manning coefficient value determined from substrate information in the sheet named “Manning coefficient” for setup the hydraulic model parameters; 2) Biological samples at which the habitat conditions were simultaneously surveyed, which mainly include biomass, water depth, velocity, and substrate in sample locations for identifying the sensitive habitat factors for multi-guilds, which are in the excel named “Biological samples”; 3) Habitat hydraulic simulation results in excel named “Habitat hydraulic simulation results”, which mainly included the distribution for the depth and velocity under different discharge rates for spatial hydraulic analysis; 4) The surveyed biomass in excel named “Surveyed biomass” for target guild (e.g., larval, Juvenile, Macro-invertebrates, and Hygrophytes) to bioverify habitat simulation model; 5) Q-WUA/WUAp% and WUAp% monthly time series included Q-WUA relationship in the sheet named “Q-WUA”, Q-WUAp% relationship in the sheet named “Q-WUAp%” and WUAp% monthly time series in the sheet named “WUAp% monthly time series”, which were used for ascertaining the threshold/optimal comprehensive environmental flows and assessing the potential ecological impacts of the hydrological change caused by the Three Gorges Dam impoundment on the suitable habitat conditions of target guilds, which are in the excel named “Q-WUA/WUAp% and WUAp% monthly time series”. The results of this paper are reproducible using the openly available data provided in this resource.