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Macau’s rapid urban development has significantly altered its natural landscapes, leading to the degradation and fragmentation of wetland ecosystems. Although diminished in size, these urban wetlands continue to deliver vital ecological services and sustain a range of biodiversity. Conventional methods for biodiversity assessment are often labor-intensive and unsuitable for regular monitoring in urban environments. This study utilized environmental DNA (eDNA) metabarcoding as a non-invasive and effective approach to survey biodiversity in 2 coastal and 7 freshwater urban wetlands in Macau. Environmental DNA was extracted from water samples collected from these nine representative wetland sites and analyzed using metabarcoding techniques to detect fish species. Three genetic markers were targeted to enhance taxonomic resolution, which included COI (mlCOIintF/LoboR1, 313 bp), 12S (miFish-U, 170 bp) and 18S (V4, 400 bp). Bioinformatic pipelines were used to process sequencing data, identify taxa, and compute biodiversity indices, providing a comprehensive snapshot of aquatic biodiversity in Macau’s urban wetlands. A total of 90 fish species were detected, including native species, migrants, and invasive taxa. Beta diversity analysis shows site and season together explained 50% of the variation in community composition (PERMANOVA R² = 0.50, p = 0.001), shaped by environmental gradients and seasonal turnover. These results highlight eDNA metabarcoding as an effective tool for biodiversity monitoring in urban wetlands and emphasize the need to preserve habitat diversity and management strategies in Macau.