Planktonic eukaryotes are important components of aquatic ecosystems, and analyses of the whole eukaryotic planktonic community composition and function have far-reaching significance for water resource management. We aimed to understand the spatiotemporal variation and drivers of eukaryotic plankton distribution in the Middle Route Project of the South-to-North Water Diversion in Henan Province, China. Specifically, we examined planktonic assemblages and water quality at five stations along the canal and another one located before the dam in March, June, September, and December 2019. High-throughput sequencing revealed that the eukaryotic plankton community was primarily composed of 53 phyla, 200 genera, and 277 species, with Cryptophyta, Ciliophora, and norank_k_Cryptophyta being the dominant phyla. Redundancy analysis of the eukaryotic community and environmental factors showed that five vital factors affecting eukaryotic plankton distribution were oxidation-reduction potential, nitrate nitrogen, pH, total phosphorus, and water flow velocity. Furthermore, the geographical distribution of eukaryotic communities was consistent with the distance decay model. Importantly, environmental selection dominantly shaped the geographical distribution of the eukaryotic community. In summary, our study elucidates the ecological response of planktonic eukaryotes by identifying the diversity and ecological distribution of planktonic eukaryotes in trans-basin diversion channels.