It is of great practical significance to ensure ecological water requirements (EWRs) for the maintenance of river health and the sustainable development of human socioeconomics. How to scientifically determine the comprehensive EWRs and estimate the uncertainty of hydro-ecological tools performed in the process of conducting remains one of the most important yet most complicated issues. In this study, the ecological water requirements framework (EWRsF) of the Lower Yellow River (LYR), which considers instream ecological base flow, survival and reproduction of indicator fish species, equilibrium of erosion and siltation and ecological function of the estuary, was constructed by integrating hydrological, hydraulic and ecological habitat methods. The framework contains three crucial components - determination of instream EWRs and estuarine EWRs, uncertainty analysis of hydro-ecological tools. For instream ecological base flow, we proposed an improved Tennant method, which took into account both seasonality and sediment transport characteristics of the LYR, and could better reflect the actual hydrological regime. For the hydrological ecological response relationship of indicator fish species, we estimated the uncertainty of the model output of River2D to improve its credibility of the simulation results. The results demonstrated that: 1) Two-grade intra-annual monthly EWRs process of suitable and minimum for four instream sections and estuary area were obtained. The flood season (June–October) is the period with the largest proportion of intra-annual instream EWRs, whereas in estuary area, is the spawning period (April–July) of dominant species. 2) The uncertainty of HSI curves directly leads to the uncertainty of model output. Although the shape and position of the WUA curve can be uncertain, it does not affect the judgment of EWRs threshold. 3) The research results can provide scientific basis for water resource management decision-making in the LYR. Additionally, the ideas also have reference significance for similar basins.