In recent decades, with the acceleration of industrialization and urbanization, human activities have interfered with natural habitats more and more frequently, leading to habitat fragmentation and habitat quality decline. Scientific simulation of the habitat fragmentation process and its impact on habitat quality at the watershed scale is of great significance to enhance biodiversity and ecosystem functioning. Taking Poyang Lake Basin as the study area, the evolution characteristics of regional habitat quality were analyzed based on the InVEST model firstly in this study. Then, the habitat fragmentation process was simulated from three characteristic dimensions of habitat area, habitat edge, and habitat isolation. Finally, the generalized additive model and geographic detector were used to explore the influence and interaction of different habitat fragmentation processes on the change of habitat quality. The results showed that: (1) the habitat quality of Poyang Lake showed a decreasing trend, from 0.7862 in 2000 to 0.7807 in 2010, and then to 0.7715 in 2020, but the overall habitat quality was better. The areas with better habitat quality were mainly distributed in the southern, northeastern and western parts of the basin. On the contrary, the areas with poorer habitat quality were mainly distributed at the junction of various sub-basins. (2) From 2000 to 2010, the three fragmentation processes of the decreased habitat area, the increase of habitat edge and the increased habitat isolation in Poyang Lake basin accounted for 34.70%, 30.15% and 4.50% of the total grid number of the study area, respectively; and accounted for 34.80%, 30.69% and 4.40% of the grid number of the total study area, respectively, from 2010 to 2020; and 40.82%, 37.50% and 5.46% of the total study area grids from 2000 to 2020, respectively. The grids with reduced habitat area were mainly concentrated in the central and northern areas of Poyang Lake basin. In contrast, the grids with increased habitat isolation were less, mainly in the central urban area of each city. (3) The habitat fragmentation process had a significant impact on habitat quality with a non-linear negative correlation. The interaction between the the three habitat fragmentation processes showed a two-way enhancement trend on habitat quality. The results can provide scientific references for the optimization of ecological pattern and the improvement of habitat quality in Poyang Lake basin.