The spatial distribution of picophytoplankton and the community structure of eukaryotic picophytoplankton were investigated in Lake Fuxian in July 2016. The results showed that average cell density of picophytoplankton (APP) was 8.58×10³ cells/mL, and picocyanobacteria (PCY) was abundant than photosynthetic picoeukaryotes (PPEs). Horizontally, picophytoplankton was more numerous in the littoral regions than in the pelagic regions. Its density was lower in the deepest region of the north lake than the deepest region of the south lake. Vertically, the abundance of picophytoplankton was the highest at the depth of 10 m, and declined with the depth. The analysis results based on variance inflation factor (VIF) and modelling indicated that turbidity, pH and total phosphorus had significant impacts on the abundance of photosynthetic picoeukaryotes. While the abundance of picocyanobacteria was mainly affected by total phosphorus. The community structure of PPEs was achieved by using the combination of flow cytometric sorting and high-throughput sequencing. In Lake Fuxian, the PPEs was mainly composed of Chrysophyceae, Bacillariophyceae, Dinophyceae, in which Chrysophcyeae was prevalent. PPEs showed more diversified in the surface water than in the deep water, being composed of Chrysophcyeae, Dinophyceae and Bacillariophyceae in the surface water, but being merely dominant by Chrysophyceae in the deep water. As the most important primary producer in oligotrophic lake, the study on the community structure of picophytoplankton would be helpful to understand the ecological structure and function of Lake Fuxian.