Abstract: This study focused on the zooplankton community in the upper reaches of the Cao’e River, a mountain stream-type river in southern Zhejiang Province. A total of 21 sampling sites were established, covering the major habitat types. Based on field sampling conducted in spring and autumn of 2024, the zooplankton community structure, niche overlap, niche breadth, and the relationships between zooplankton and environmental factors were systematically analyzed. The aim was to reveal the response mechanisms of zooplankton communities to environmental changes and to provide a scientific basis for maintaining ecological balance and supporting management decisions in the Cao’e River.The results showed that a total of 81 zooplankton species from four groups were recorded in the study area, including protozoans, rotifers, cladocerans, and copepods. The zooplankton community composition was dominated by protozoans and rotifers. Species density was also mainly contributed by protozoans and rotifers, whereas biomass was dominated by copepods. Zooplankton density and biomass in autumn were significantly higher than those in spring, and both density and biomass were higher in lake/reservoir-type waters. Regarding environmental factors, water temperature, dissolved oxygen, chlorophyll, ammonia nitrogen, and chemical oxygen demand were generally higher in autumn than in spring, while total nitrogen was higher in spring than in autumn. Temperature, chlorophyll a, transparency, total nitrogen, and total phosphorus had significant effects on zooplankton community structure and niche characteristics. In spring, zooplankton community density was mainly affected by transparency and chlorophyll a, whereas in autumn it was mainly driven by chlorophyll a and total phosphorus. The zooplankton indices in spring and autumn indicated that the overall water quality was at a moderate pollution level. Species diversity in autumn was slightly better than in spring, but species richness decreased in autumn, while evenness remained relatively stable. Niche breadth analysis showed that zooplankton species could be classified into broad-niche, intermediate-niche, and narrow-niche species. Broad-niche species, such as nauplii (Nauplius), exhibited strong environmental adaptability, while narrow-niche species, such as Difflugia acuminata, had more specific environmental requirements. Niche overlap analysis indicated that interspecific competition among zooplankton was more intense in autumn. The alternating rapid-flow and still-water habitats in the upper reaches of the Cao’e River provide diverse habitats for zooplankton, promoting the maintenance of biodiversity and reflecting the complexity, diversity, and stability of this ecosystem. Therefore, future work should not only include long-term monitoring of the upper Cao’e River, but also integrate conservation measures to maintain the dynamic balance of the ‘river-lake-stream’ environment and promote a healthy ecological cycle.