As major primary producers in aquatic systems, the diversity and community composition dynamics of photosynthetic picoeukaryotes (PPEs) have been investigated in recent years. Here, we explored the 3-day diurnal succession of the PPE community in a highly eutrophic lake in early spring using a combination of flow cytometric sorting and high-throughput sequencing. Our results showed that the PPEs were the dominant group of autotrophic picophytoplankton, although they had relatively low diversity and were dominated by the Stephanodiscaceae family in early spring in Lake Taihu. Furthermore, PPE abundance was significantly higher at night than during the day, probably due to their high specific rate of carbon uptake during the day and rapid proliferation at night. Several environmental variables had a significant influence on the PPE community composition, and the daily effect was more important than the diurnal effect when shaping the PPE community in Lake Taihu at a temporal scale of 3 days. Furthermore, based on the variation partitioning analysis (VPA), the relative importance of abiotic factors (deterministic processes) to short-term succession was low, explaining only 20.44% of the PPE community variation. We therefore conclude that stochastic processes determined PPE community assembly over a short temporal scale based on a neutral community model (NCM).