<p>The Guangdong-Hong Kong-Macao Greater Bay Area (GBA) is a critical region in the Pearl River Delta along the South China coast and has a remarkably diverse seed plant species. However, factors such as rapid urbanization and climate change are increasingly impacting the resilience of the Greater Bay Area’s ecosystems. While morphology identification has many drawbacks such as slow process, incorrect identifications, and unreliable for distinguishing species at the growth stage; DNA barcoding has become a valuable tool in plant taxonomy by effectively overcoming many limitations of traditional methods. In this study, we constructed a comprehensive DNA barcoding database for native seed plants in the GBA using three barcodes (<i>matK, rbcL</i>, and ITS2). A total of 2864 native species from 1117 genera and 192 families were represented, of which 695 individuals from 516 species that were newly generated. This study enhances sustainable management and accurate identification of species, facilitates research on plant evolution and ecology, and supporting biodiversity monitoring and conservation efforts within the Greater Bay Area.</p>