Anthropogenic habitat alteration interferes the natural aquatic habitats and the system’s hydrodynamics in the Yangtze River floodplain lakes, resulting in a serious decline in freshwater biodiversity. Zooplankton communities possess major position in freshwater ecosystems, which play essential parts in maintaining biological balance of freshwater habitats. Knowledge of processes and mechanisms for affecting variations in abundance, biomass, and diversity of zooplankton is important for maintaining biological balance of freshwater ecosystems. Here, we analyzed that the temporal and spatial changes in the structure of zooplankton community and their temporal and spatial variations respond to changes in environmental factors in the middle reach of Yangtze River floodplain lakes. The results showed that zooplankton samples were classified into 128 species, and Rotifera was the most common taxa. Significant seasonal differences were found among the abundance and diversity of zooplankton. Similarly, we also found significant seasonal differences among the biomass of zooplankton functional groups. The spatial turnover component was the main contributor to the β diversity pattern, which indicated that study areas should establish habitat restoration areas to restore regional biodiversity. The NMDS plot showed that the structure of zooplankton community exhibited significant seasonal changes, where the community structure was correlated with pH, water temperature, water depth, salinity, total nitrogen, chlorophyll-a, and total phosphorus based on RDA. This study highlights that it is very important to ensure the floodplain ecosystem’s original state of functionality for maintaining the regional diversity of the ecosystem as a whole.