The middle and lower reaches of the Yangtze River are not only the areas with high biodiversity levels in China, but also the areas with the fastest economic development rate. The continuous change in regional land use has a profound impact on the structure and quality of the regional ecosystem. Therefore, to reveal the impacts of land use changes on habitat quality, this study used the InVEST model and geographically weighted regression (GWR) model to estimate the habitat quality quantitatively and analyze the spatiotemporal distribution characteristics of the habitat quality based on the land use data in the years of 1990, 2000, 2010, and 2020 in the area of the middle and lower reaches of the Yangtze River. The results showed that the main land use types in the study area were paddy land and forest land, and the area of the two types accounted for more than 65% of the whole area. From 1990 to 2020, construction land expanded by 452.68 km2 and was concentrated along the Yangtze River and in the downtown areas of Maanshan City, Wuhu City, and Tongling City, while the farmland in the study area was reduced by 1227.92 km2. From the perspective of habitat quality, the area proportion of the lowest grade of habitat quality increased from 4.85% in 1990 to 8.47% in 2020. The average values of habitat quality were 0.5918 in 1990, 0.5902 in 2000, 0.5850 in 2010, and 0.5814 in 2020, which showed a trend of continuous decline in the average value of habitat quality in the study area. The areas with low values of habitat quality were mainly concentrated along the Yangtze River. From 2010 to 2020, the impact of paddy land and dry land on habitat quality showed a two-level differentiation trend, and the lowest regression coefficients were -0.4431 and -0.121, respectively. The impact of forest land on habitat quality was mainly positive, with the highest value of the regression coefficient of 0.657. The impact of construction land on habitat quality was mainly negative, and it was concentrated along the Yangtze River and south of it, with the lowest value of -1.0625.