Fish in the Yong River basin have been significantly impacted by pollution, habitat modification and overfishing. In order to facilitate the recovery of freshwater biodiversity, a fishing ban has been implemented in the Yong River basin since 2022. However, the high level of human activity along the waterways of the Yong River basin poses a significant challenge to the monitoring of fish biodiversity using established methods. It is essential, therefore, to have effective monitoring tools to assess the effectiveness of the fishing ban policy. In this study, environmental DNA (eDNA) metabarcoding was employed to assess fish species biodiversity across five river sections of the Yong River basin in October 2022. Additionally, we checked the results against fishing gear (drift gillnets and ground cages) surveys and historical records. A total of twenty-two fish species were found from eDNA metabarcoding, fourteen species were found via fishing, and only two species were found from both methods. The most dominant fish species identified by eDNA was Alburnus mossulensis, present in all river sections. However, a decline in species biodiversity was observed in the whole Yong River basin, with a significant difference in community composition across the Upper Yao River (UYAR) and Yong River (YOR). Moreover, eDNA detected fish and potentially invasive species consistent with their geographic distribution. Overall, the results of this study provide a snapshot of the current fish biodiversity in the Yong River basin, offering baseline data for future evaluations of fishing ban policies in this ecosystem.