Accurate detection of the composition and dynamics of harmful algal bloom (HAB) species is critical for studying the mechanisms of HAB formation and for developing means for predicting the occurrences of HABs. Jiaozhou Bay is an epitome of China’s coastal ecosystem and an ideal site for HAB research with the accumulation of decades of historical investigation records. Nevertheless, most of these earlier studies on phytoplankton communities applied primarily morphology-based approaches with limited resolution in phytoplankton species identification, especially for those with small-sized cells and for cryptic species. Through analyzing samples collected at 12 spatially isolated locations using metabarcoding methods, 89 phytoplankton species, including 34 Bacillariophyta, 25 Dinoflagellata, 7 Cryptophyta, 11 Chlorophyta, 8 Ochrophyta and 2 Haptophyta species were detected. Of those, 70 species had never been reported in Jiaozhou Bay in the previous expedition investigations, demonstrating the strength of the metabarcoding analysis approach. The distribution of many algal species demonstrated unique patterns, which were likely influenced by interactions among phytoplankton species or by predation by groups such as Ciliophora and Cercozoa, in addition to environmental factors such as temperature and nutritional conditions. Among these algal species, 28 were annotated as HAB species, among which 13 were reported for the very first time in Jiaozhou Bay including a mixtotrophic dianoflagellate Heterocapsa rotundata and a chain-forming diatom Skeletonema marinoi, both ranked among the top 10 most abundant ASVs. The present study represents a first attempt to study HAB species and other phytoplankton species in Jiaozhou Bay using the metabarcoding approach, which revealed substantially more algal species in Jiaozhou Bay than previously identified and sets a solid foundation for further research on the mechanisms of HAB formation.