This study investigated zooplankton distribution in Lake Dianchi, focusing on river mouths and their ecological conditions, using environmental DNA (eDNA) technology. It analyzed zooplankton diversity during the dry and wet seasons, employing alpha and beta diversity indices to assess community differences. A total of 24 species from 22 genera and 17 families across two phyla were identified, with Arthropoda (54%) and Rotifera (46%) dominating. Daphniaceae made up 20.8% of arthropods. β-diversity analysis showed a 37% difference between seasons and significant differences between estuaries: 61.4% in the dry season and 42.1% in the wet season. Eucyclops macruroides dominated peri-urban estuaries, while Arctodiaptomus altissimus pectinatus prevailed in urban, agricultural, and central lake areas. Redundancy analysis identified total nitrogen (TN) as the main factor affecting zooplankton diversity, with the highest TN levels in suburban estuaries. The findings provide insights for ecological restoration efforts in Lake Dianchi estuaries.