Marine biodiversity is increasingly threatened by overfishing and marine pollution. A comprehensive understanding of the biodiversity in the Yellow and Bohai Seas is essential for resource conservation. This study conducted a large-scale survey of fishery resources in the Yellow and Bohai Seas to investigate fish community structure and diversity using eDNA technology. The results showed that 57 and 65 fish species were recorded in spring and summer, respectively, with 46 species identified in both seasons. A total of nine dominant species were identified in spring, mainly belonging to Perciformes, while seven dominant species were identified in summer, primarily from Clupeiformes. In different regions, no significant differences in fish α diversity were observed between spring and summer. However, significant seasonal differences were found in both α and β diversity. At the spatial level, fish species in the Yellow and Bohai Seas occupied similar ecological niches. However, at the temporal level, there are certain differences in the ecological niches of fish communities between seasons. Phylogenetic diversity showed no significant differences at the spatial scale, yet notable seasonal variations were observed at the temporal scale, with spring (661.46) significantly exceeding summer (540.30). The Mantel test indicated that nitrate and salinity were key environmental factors influencing α diversity. RDA analysis revealed that the dominant species in spring were mainly influenced by chlorophyll-a and water temperature, while those in summer were mainly affected by water depth, nitrite, water temperature, and salinity.