Phytoplankton is critical to river ecosystems. These organisms are sensitive to streamflow and water quality changes and, therefore, used to determine stability of river ecosystems, especially in regulated rivers. However, exactly how such disturbances alter spatial distribution of phytoplankton remains unclear, particularly during different seasons. A thorough understanding of these mechanisms is required to better analyze impact of environmental factors on regulated rivers. Given this, phytoplankton communities, streamflow, and water quality factors were assessed in areas sampled four times from 2015 to 2016 in upper and middle Huai River Basin. Biodiversity indices, as well as cluster and rank analyses, were used to (1) determine phytoplankton composition and distribution and (2) clarify impacts of key streamflow and water quality factors on such communities. It was found phytoplankton composition deteriorated over time, with phyla number decreasing from six to three. Moreover, proportion of Bacillariophyta increased from 51.83% to 68.13%. Phytoplankton in three regions, upstream region (Shannon-Wiener index 1.39–2.95), midstream region (0.70–4.55), and downstream region (0.22 to 2.97), were spatially clustered. The most impact factors impacting variation in composition and distribution were water quality factors and then hydrological factors. Of these, the most important factors in wet seasons were total nitrogen and maximum runoff, while ammonia nitrogen and low flow discharge were the most important factors during dry seasons. Streamflow and water quality contributed the most in midstream region, which was significantly affected by numbers of high and low flow. Contributions of these factors to downstream region were the strongest during dry seasons, which were significantly affected by numbers of low flow. Collectively, these results reveal significant impact of streamflow and water quality factors on phytoplankton deterioration in upper and middle Huai River Basin. Critically, this study provides scientific and technological support for increased biomonitoring and ecohydrological studies in regulated river basins.