Abstract Background The drastic change in an ecosystem as a threshold phenomenon caused by abrupt changes in environmental conditions is a focus of current ecological research. However, the study of ecological thresholds has generally been limited to estimating the threshold values of single factors. Using eDNA metabarcoding technology, we collected zooplankton data from Baiyangdian Lake, the largest freshwater lake in the North China Plain, to explore the zooplankton community distribution characteristics and the relevant environmental factors. We used Threshold Indicator Taxa Analysis (TITAN) to determine the thresholds of key environmental factors and to identify the factors influencing biological diversity. Results By comparing previous studies, we found that the zooplankton community composition based on eDNA metabarcoding was similar to that based on morphological methods, and that the data could be used to estimate ecological thresholds and assess risk conditions. Temperature (T), electrical conductivity (EC), and turbidity were the major environmental factors affecting the zooplankton community structure. The composition and structure of zooplankton communities in rivers and lakes were significantly different due to the influence of specific environmental factors. The results of TITAN analysis showed that there were different indicator species for T and EC in rivers and lakes. The protection thresholds of zooplankton in rivers were T = 19.0 °C and EC = 795 μS/cm, whereas the protection thresholds of zooplankton in lakes were T = 14.3 °C and EC = 1920 μS/cm. The overall values for the Baiyangdian watershed were T = 15.5 °C and EC = 1073 μS/cm. Compared with the field monitoring results, approximately 50% of the water quality index values at the sampling points in the Baiyangdian watershed exceeded the negative response threshold, indicating that Baiyangdian Lake was disturbed. Conclusions The validity of eDNA technology in biodiversity analysis was confirmed by the zooplankton community data from Baiyangdian Lake. The ecological thresholds derived by combining eDNA technology with Threshold Indicator Taxa Analysis (TITAN) are beneficial to the biological conservation of the region.