As the biggest basin in China, the impact of urbanization on habitat quality in the Yangtze River Basin show obvious gradient differences. Revealing the gradient differences of the impact of urbanization on habitat quality is crucial for the Yangtze River protection and the development of differentiated control measures for habitat restoration. However, few previous studies still reveal gradient differences in the impacts of urbanization on habitat quality in basin regions, which has brought about some knowledge gaps and limited the development and improvement of related policies. To this end, this study established 100km and 300km buffer zones along the main stream of the Yangtze River, and set buffer zones within 100km as the inner basin, buffer zones between 100 and 300km as the outer basin, and the rest of the area as the border basin as sub-regional divisions. Then, this study introduced the Dagum Gini coefficient, kernel density estimation, and spatial statistics to analyze the regional differences in urbanization level and habitat quality among different sub-regions, respectively, and introduced a set of spatial regression models to analyze the impact of urbanization on habitat quality in different sub-regions to reveal the gradient differences in the impact of urbanization on habitat quality. The results showed that: (1) the habitat quality in the Yangtze River Basin decreased and then increased along the buffer gradient and deteriorated over time in different gradient intervals, while the urbanization level continued to decrease along the buffer gradient but increased dramatically over time in different gradient intervals. (2) Intra-regional and inter-regional differences in habitat quality between different gradient intervals have been increasing, with the intensity of transvariation being the main source of regional differences, and trends in habitat quality shifts between different gradient intervals were also highly variable. (3) The urbanization level was mainly negatively correlated with habitat quality, and the negative impact of urbanization on habitat quality increased along the buffer gradient but weakened over time. Based on the results of the study, we propose that the process of biodiversity conservation in the Yangtze River Basin should be tailored to the specific natural conditions of the different gradient intervals, as well as the mode and level of urbanization and socioeconomic development goals. The implementation of reasonable habitat management and restoration measures can better alleviate the resource and environmental pressures brought about by urbanization. Meanwhile, this study may also provide a case study for researching gradient differences in the resource and environmental effects of urbanization in other basin areas.