Abstract: Human activities and global climate change are posing ongoing threats to fish diversity, leading to imbalances in community structure and a decline in ecosystem functions, thereby endangering the sustainable utilization of fishery resources. Under the combined pressure of multiple stressors, accurately understanding the spatiotemporal dynamics of fish diversity has become a critical issue that urgently needs to be addressed for ecological conservation and resource management. To validate the suitability of environmental DNA (eDNA) metabarcoding for monitoring fish diversity in the Yangtze River Estuary, and to reveal the spatiotemporal dynamics of fish community structure in this area, this study employed eDNA metabarcoding technology to investigate the fish biodiversity in the Yangtze River Estuary and its adjacent waters during the spring and autumn seasons of 2022 and 2024. Through filtration of seawater samples, DNA extraction, amplification with MiFish primers, high-throughput sequencing, and a stringent bioinformatics pipeline coupled with taxonomic annotation against the MitoFish database. This study obtained 17,477,708 raw sequences, and after quality control, 16,690,083 high-quality sequences were obtained, with sequencing quality values (Q30) all above 85%. A total of 219 fish species were identified, belonging to 2 classes, 21 orders, and 87 families, with a matching rate of 84.93% compared to historical literature records. In terms of species composition, Perciformes was the dominant group (102 species, accounting for 46.58%). Analysis of alpha diversity indices (Shannon, Simpson, Pielou) revealed significant seasonal variations in fish community structure, with species richness and diversity indices in spring 2024 significantly lower than those in spring 2022. Beta diversity analysis (NMDS) further elucidated the seasonal differentiation pattern of the community structure. The composition of dominant species indicated a community shift towards miniaturization and lower trophic levels. This study, utilizing environmental DNA (eDNA) metabarcoding, has revealed the seasonal dynamics and interannual variation trends of fish community structure in the Yangtze River Estuary. It confirms the effectiveness of this technology for monitoring fish diversity in this region, providing critical data to support biodiversity conservation and adaptive management in the area.