Water and sediment have always been closely tied in aquatic systems. However, little information regarding the full extent of microeukaryotic composition in both the two habitats did we know especially in estuaries. In the present study, the microeukaryotic abundance, diversity, composition, and their response to environmental factors between sediment and water in the Yellow River Estuary (YRE) were investigated. The microeukaryotic 18S rRNA gene abundance ranged from 1.03 × 106 to 5.48 × 107 copies/g dry for sediment, and 3.01 × 104 to 1.25 × 106 copies/mL for water. The distribution patterns of eukaryotic microorganisms could be clustered into two different branches. And the compositions of microeukaryotes in the two habitats were distinct obviously. Metazoa, Fungi, Streptophyta, Ochrophyta, Cercozoa, and Dinophyta were more abundant in sediment. The dominant phyla in water were Dinophyta, followed by Metazoa, Ochrophyta, Cryptophyta, Chloroplyta, Cercozoa, Fungi, Katablepharidophyta, Choanoflagellida, and Haptophyta. Interestingly, the eukaryotic microorganisms detected in sediment were much less sensitive to environmental variables compared with water. Furthermore, their potential co-occurrence networks in particular were also discovered in the present study. As such, we have provided baseline data to support further research on estuarine microeukaryotes in both sediment and water, which was useful for guiding the practical application of ecosystem management and biodiversity protection.