Picoeukaryotes (< 2–3 μm) are major oceanic primary producers and play a crucial role for the functioning of marine ecosystems. However, the community structure of such communities remains poorly understood, especially in the East China Sea (ECS). We investigated the seasonal variations of abundance and diversity of picoeukaryotes, and recorded environmental variables, along a transect in Zhoushan Fishing Ground. High-throughput sequencing was used for sequencing the V4–V5 variable region within the 18S rRNA gene to analyze genetic diversity and relative abundance of picoeukaryotes. A total of 40 phyla, 68 classes, 99 orders, 126 families, and 140 genera were observed. The estimated richness and diversity indices were both higher in each season. The composition and dominant picoeukaryotes changed significantly at the class level with the seasons alternating. Basidiomycota, Ciliophora, Ascomycota and Cryptomonadales were observed throughout the year. The Linear Discriminant Analysis (LDA) revealed the predominant discriminant taxa of four seasonal groups. Redundancy analysis (RDA) showed that NO 2 − and salinity played very important roles to picoeukaryotes for all the spring samples and DS5 sample in winter, PO 4 3− and pH affected mostly picoeukaryotes for winter samples and autumn samples.