Abstract: The amphioxus, a protected species, is regarded as a “living fossil” for studying the origin and evolution of vertebrates and possesses significant academic value. To investigate the diversity and abundance of eukaryotic community within the digestive tract of amphioxus inhabiting Xiamen Bay, this study analyzed the composition and abundance of eukaryotic organisms in the digestive tract of amphioxus using high-throughput sequencing targeting the V4 region of the 18S rDNA. SILVA database taxonomic annotation revealed a diverse eukaryotic community, with plant components accounting for over 50% of the total sequences. The relative abundance followed a descending order: Chlorophyta, Phragmoplastophyta, Dinoflagellata, fungi, Vertebrata, protozoa, and Bacillariophyta. The eukaryotic composition in the digestive tract of amphioxus showed no significant spatial variation between the Huangcuo and Nanxian-shibaxian sea areas. However, significant shifts were observed between different body length groups. Annotations against the PR2 database further identified that the phytoplankton in the amphioxus digestive tract was primarily composed of Chlorophyta, Dinoflagellata, and Bacillariophyta. DNA metabarcoding recovered abundant information on eukaryotic diversity in the amphioxus digestive tract.