To systematically compare the differences in fish community composition and diversity patterns, and to evaluate the applicability of environmental DNA (eDNA) metabarcoding technology for monitoring estuarine fishery ecosystems, this study collected water samples from nine stations in each of the two major estuaries during the spring of 2025. eDNA was extracted, and a fragment of the fish 12S rRNA gene was amplified and subjected to high-throughput sequencing. After quality control and taxonomic annotation of the sequencing results, species composition and diversity were assessed. The results revealed a total of 57 fish species identified across both estuarine regions, with the number of fish species in the Yangtze River Estuary (43 species) being significantly higher than that in the Yellow River Estuary (30 species). The community structure in the Yellow River Estuary showed a pattern dominated by a single dominant species (Trachurus japonicus), whereas the Yangtze River Estuary was co-dominated by multiple species such as Coilia mystus, T. japonicus, and Engraulis japonicus, exhibiting a more even distribution. Alpha diversity indices indicated that species richness, the Shannon index, and Pielou's evenness were all significantly higher in the Yangtze River Estuary than in the Yellow River Estuary. Non-metric multidimensional scaling (NMDS) analysis showed large differences in community composition among stations and high spatial heterogeneity within the Yellow River Estuary, while stations in the Yangtze River Estuary exhibited higher similarity. This study demonstrates that fish diversity in the Yangtze River Estuary is higher than in the Yellow River Estuary during spring, with significant differences in dominant species patterns and spatial heterogeneity between the two, which may stem from differences in their hydrological environments and habitat complexity. The findings provide a new data perspective for the baseline assessment and management of fishery resources in the Yellow River and Yangtze River estuaries, and offer an empirical case study and methodological reference for the application of eDNA technology in the routine monitoring of biodiversity in China's coastal waters.