It is important to assess the aquatic ecosystem in large-scale basins when considering complicated influential factors of anthropogenic activities on these ecosystems. Watershed management needs more information on ecological function and services in large regions. Spatial units are particularly important for the watershed management. Eco-regions are effective solutions to integrate the terrestrial and aquatic ecosystems. Many studies have been focused on several local watersheds. However, few studies have been implemented on the correlations between the terrestrial and aquatic ecosystems in large-scale watersheds. For a case study, we selected the Haihe River Basin to investigate the methods and indicators of the aquatic eco-regions. This study was conducted to assess the river ecosystem in the Haihe River Basin by sampling 249 river sites during the pre-and post-rainy seasons from 2013 to 2015. Water sampling and sample preservation were conducted in accordance with Chinese national experimental standards. Macroinvertebrates were collected from each sampling site at the same time as water sampling. The sampling method for macroinvertebrates was based on rapid bioassessment protocols. After identifying the spatial characteristics of terrestrial and fluvial landscapes, we proposed some guidelines and criteria for regionalizing the terrestrial and aquatic ecosystems in the Haihe River Basin. Three levels of eco-regions were used to differentiate the terrestrial and aquatic ecosystems. The first and second levels of eco-regions were identified using "top-down" methods. The regionalization indicators included climate, topography, vegetation, and soil. Six first-level and 16 second-level eco-regions were regionalized in this region. These eco-regions could be used to guide land use planning and ecological management. The third-level eco-regions were identified using "bottom-up" methods. The regionalization indicators included water consumption of production and living, pollutant discharge, watershed geometry, landscape pattern, land use, and river types. The resulting 73 eco-regions were obtained by coupling the administrative boundaries and second-level eco-regions. Finally, the eco-regions were evaluated by field survey data including water quality, aquatic vegetation, and aquatic communities. The significance level of the correlations exhibited the influence of terrestrial indicators on the aquatic ecosystem. The third-level eco-regions can be used to direct the land use planning for specific socioeconomic activities, water resources, and pollutant loads. These eco-regions identify priority areas for ecological restoration and aquatic life protection. This study shows how different data sets can be used to regionalize eco-regions and what specific factors should be included in the indicator system. The results can potentially be used to improve the ecological management in the Haihe River Basin and can be useful to regionalize the ecosystems in other large watersheds.