Poyang Lake plays a significant role in maintaining and replenishing the macrozoobenthos biodiversity in the middle Yangtze River. However, due to human activities and natural factors, the habitat of Poyang Lake has been seriously degraded, resulting in a decline in macrozoobenthos biodiversity. Here, we analyzed the effect of human activity and environmental elements change on the diversity of macrozoobenthos based on a systematic investigation of Poyang Lake Basin in 2016–2017. The current species richness, density, and biomass of macrozoobenthos were lower than those in the historical period. At the same time, the community structure of the macrozoobenthos assemblage exhibits significant temporal and spatial differences. In addition, the spatial turnover component was the main contribution to beta diversity, which indicated that a number of protected areas would be necessary to conserve the biodiversity of macrozoobenthos. Water depth, dissolved oxygen, water velocity, and chlorophyll‐a were significantly correlated with macrozoobenthos distributions and assemblage structure based on RDA. These results indicated that human activities have seriously destroyed the macrozoobenthos habitat and led to the decline in macrozoobenthos diversity. Therefore, habitat restoration and the conservation of macrozoobenthos have become urgent in Poyang Lake Basin, and an integrated management plan should be developed and effectively implemented.