We report an Illumina high-throughput sequencing protocol of eukaryotic microbes in the world’s deepest marine blue hole, Sansha Yongle Blue Hole, Xisha, South China Sea. The variable V9 region of small subunit (SSU) rDNA, was sequenced using this approach from the waters of blue hole and outer reef slope. 917,771 unique eukaryotic 18S rRNA gene sequences and 6093 operational taxonomic units (OTUs) were identified. Significant differences in the eukaryotic composition were observed between the blue hole and outer reef slope, and the richness in the blue hole was much higher than that in the outer reef slope. The richness and diversity of eukaryotes in the blue hole were both lowest at 60 m and highest at 100 m depth. Eukaryotic microalgae assemblages dominated by Dinophyceae were the most abundant in the 10–20 m water column in the hole. Fauna was the main group at and below a depth of 60 m, where Araneae and Cyclopoida were dominant in the 60 m and 80 m water layer, respectively. There was a large number of Entoprocta at a depth of 180 m in the hole, where little oxygen was detected. Turbidity and nitrite concentration had a significant effect on the eukaryote community structure (p < 0.01).

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