In order to investigate the spatiotemporal pattern of phytoplankton communities and the determining factors in the channel of the Middle Route of South-to-North Water Diversion Project, seasonal samples of phytoplankton and aquatic from 11 different sample stations were collected and analyzed from 2018 to 2019. Phytoplankton species were detected as 7 Phylum, 145 species, with the largest number of taxa belonging to Bacillariophyta. The dominant taxa were Achnanthidum minutissimum, Cymbella sp., Coelastrum sphaericum, Chlamydomonas sp., etc. (Y>0.02). The chlorophyll-a concentration was (4.09 ±2.89) μg/L, and the cell density was (6.79±7.15)×10⁶ cells/L, with the maximum cell density in summer. Chlorophyta was dominant in summer, but Bacillariophyta were prevalent in other seasons. There existed a gradually increase in chlorophyll-a concentration and cell density along the channel, and there were significant differences before and after the Yellow River Tunnel Project; Meanwhile, the results of redundancy and multiple linear regression analysis for the relationship between the phytoplankton and environment showed that water temperature and nutrient were the key factors, and the second was water flow and water velocity; NMDS and cluster analysis indicated the phytoplankton community-owned obvious spatiotemporal heterogeneity in the long-distance water transportation, and the seasonal changes showed much more pronounced effect on the phytoplankton community compositions than spatial variation. In view of the determining role of flow rate and flow velocity on phytoplankton structure, more attention should be paid to the ecological scheduling regulation of phytoplankton community in the management of water quality safety and stability.