Understanding the evolution of the rural-urban interface (RUI) and its dynamic ecological consequences is of great theoretical and practical significance for urban-rural integration and management. In this study, Nanjing is taken as an example, and the evolution of the RUI in 1990, 2000, 2010, and 2020 and its effects on the ecological landscape structure and function are investigated. We first developed a discriminant matrix to identify the RUI during different periods based on land use maps and nighttime light data. The response of ecological landscape structure and function to the RUI evolution was analyzed using landscape metrics and ecosystem services value (ESV). The results show that the RUI experienced significant inter-transfer with rural and urban areas, and three evolution patterns were concluded, i.e., edge developing, rural industrializing, and outlying. The evolution of the RUI has led to landscape fragmentation and enhanced ecological land connectivity at the city scale. At the community/village scale, the shape of farmland became more regular with the evolution of the RUI. The transformation from rural to RUI and the maintenance of RUI would increase landscape fragmentation, richness, and the intertwining of artificial and natural land. The overall ecological function in terms of ESV usually decreased with the evolution of the RUI, except when the area of ecological land increased significantly. We believe that urban expansion, planning and protection policies, and industrial orientations are major factors facilitating the impacts of the RUI evolution on ecological landscape structure and function. At last, we gave suggestions of landscape management approaches and ecosystem services trade-off/synergy for the improvement of ecological landscape structure and function.