Aquatic ecoregions were increasingly used as spatial units for aquatic ecosystem management at the watershed scale. In this paper, the principle of including land area, comprehensiveness and dominance, conjugation and hierarchy were selected as regionalizing principles. Elevation and drainage density were selected as the regionalizing indicators for the delineation of level I aquatic ecoregions, and percent of construction land area, percent of cultivated land area, soil type and slope for the level II. Under the support of GIS technology, the spatial distribution maps of the two indicators for level I and the four indicators for level II aquatic ecoregion delineation were generated from the raster data based on the 1,107 subwatersheds. River subbasin taxonomy concept, two-step spatial clustering analysis approach and manual-assisted method were used to regionalize aquatic ecosystems in the Taihu Lake watershed. Then the Taihu Lake watershed was divided into two level I aquatic ecoregions, including Ecoregion I1 and Ecoregion I2, and five level II aquatic subecoregions, including Subecoregion II11, Subecoregion II12, Subecoregion II21, Subecoregion II22 and Subecoregion II23. Moreover, the characteristics of the two level I aquatic ecoregions and five level II aquatic subecoregions in the Taihu Lake watershed were summarized, showing that there were significant differences in topography, socio-economic development, water quality and aquatic ecology, etc. The results of quantitative comparison of aquatic life also indicated that the dominant species of fish, benthic density, biomass, dominant species, Shannon-Wiener diversity index, Margalef species richness index, Pielou evenness index and ecological dominance showed great spatial variability between the two level I aquatic ecoregions and five level II aquatic subecoregions. It reflected the spatial heterogeneities and the uneven natures of aquatic ecosystems in the Taihu Lake watershed.