As China rapidly urbanizes, land resources tend to deplete. This paper aims to identify and propose a resolution of land use conflicts to promote sustainable land use and coordinate the interaction between humans and the environment in urban areas. The methodology of land use conflict assessment within spatial zoning of the Su–Xi–Chang region was evaluated. Taking into consideration the intensity of human activities and the background condition of the natural environment, we divided the study area into a few regions. Furthermore, we developed a methodology by calculating landscape complexity, fragility, and stability based on spatial zoning so as to derive the spatio-temporal characteristics of the land use conflict index (LUCI) in the Su–Xi–Chang region. The results indicate the following: (1) According to the urban spatial attribute index (USAI) statistics, we delineate the core, fringe, and suburban zones of the Su–Xi–Chang region, which accounted for 9.61%, 11.06%, and 79.33% of urban agglomerations respectively; (2) from 1990 to 2018, the fragility index (FI) and LUCI showed S-shaped curves, while the complexity (CI) and the stability indices (SI) exhibited minor fluctuations in the fringe and suburb zones; and (3) intensive and severe conflict is concentrated in core areas due to quite intense human activities and in fringe areas due to an increased interaction between humans and land, while moderate conflict is mainly found in rural and suburban areas that pose as a natural environmental space. The results can serve as a theoretical framework for an effective identification of the LUCI in an urban agglomeration and coordination of the optimal allocation of regional land resources.