Context: The impact of construction land expansion on regional landscape sustainability received significant attention, but the habitat isolation caused by such expansion across the urban-rural continuum calls for a closer examination. Objective: This study aims to use the ecological network approach to assess the isolation effect imposed by urban areas and rural settlements on habitat patches in the Nanjing Metropolitan Area during 2000, 2010, and 2020. Methods: We first extracted the habitat patches by applying morphological spatial pattern analysis (MSPA), then identified the ecological networks based on the comprehensive resistance surface and circuit theory. Finally, we constructed two indices, the isolation effect index (IEI) and isolation degree (ID), and evaluated the differential contributions of urban areas and rural settlements to habitat isolation. Results: Our results showed a total of 129 habitat patches within our study area. These patches were linked by 188, 186, and 183 ecological corridors in the years 2000, 2010, and 2020, respectively. Further analysis revealed that habitat patches were strongly isolated by the expansion of construction land and increasing human activities. Remarkably, both urban areas and rural settlements played pivotal roles in exacerbating this isolation, with urban areas showing a striking surge in their isolation impact, while rural settlements continued to be the predominant driver of habitat isolation. Conclusions: Sustainable landscape planning should consider how land uses may cause habitat isolation. Our study utilizes the ecological network approach to evaluate habitat isolation and introduces applicable indicators for estimating the isolation effects attributed to construction land expansion. Our findings hold significant implications for informing landscape planning and shaping ecological conservation policies.