As the negative impacts of human activities on natural ecosystems continue to intensify, non-invasive biodiversity assessment is becoming more and more important. The purpose of this study was to study fish diversity in Nanchang section of the lower reaches of Ganjiang River using environmental DNA metabarcoding technology. It compared the species composition and diversity of fish environmental DNA information from different seasons (spring, summer, autumn and winter), different water layers (upper, middle and lower) and different sampling locations (nearshore and offshore). The results showed that 114 species of fish were detected, among which 83 species were recorded in history. The diversity and composition of fish environmental DNA information in different seasons showed significant differences. The number of fish species detected in upper water was significantly higher than that in middle water and lower water, respectively. Most of the fish detected in the middle and lower waters were also detected in the upper water, respectively. There were no significant differences in diversity and composition of fish environmental DNA information in upper water, middle water and lower water. More fish species were detected near shore than offshore. There was no significant difference in fish diversity index, but significant difference in community structure. RDA analysis showed that fish environmental DNA in the lower reaches of Ganjiang River were greatly affected by temperature and pH. This study can provide baseline data for the investigation of fish resources based on environmental DNA metabarcoding, and a basis for the implementation of different sampling strategies for the subsequent monitoring of environmental DNA metabarcoding, in Ganjiang River. This study can also provide a technical reference for studying fish diversity in flowing water system using environmental DNA metabarcoding technology, and a basis for standardization process of application of environmental DNA metabarcoding technology.