Constructing ecological networks in urban areas improves ecosystem stability and biodiversity protection. However, most studies focus on optimizing ecological environments through objective assessments, often neglecting species diversity. This study developed a biodiversity grading framework for the Jianghan Plain using species observation and ecosystem diversity data. Supported by ArcGIS, ecological sources were identified via MSPA and graded using the Guidelines and MaxEnt model. The MCR model was used to simulate connectivity barriers between ecological sources and calculate the minimum cumulative resistance distance, thereby generating corridors and ultimately constructing a hierarchical biodiversity conservation network for the Jianghan Plain. Our findings indicated the following: (1) The Jianghan Plain hosts 21 major ecological sources, primarily natural water bodies at the plain’s edge, which can be classified into five primary and 16 secondary sources based on biodiversity grades. (2) The recessive corridors, comprising 10 primary and 95 secondary ones, are mainly concentrated in the central Jianghan Plain, with primary corridors located centrally and westward, characterized by a large overall span. (3) Changhu Lake and Honghu Lake, two critical water bodies with high-quality habitats and significant biodiversity, were identified as key ecological nodes from the ecological sources, bridging and guiding the central and southern corridors. (4) Based on the ecological network distribution and key nodes and corridors, a “three zones, three belts, and two points” strategy was proposed for optimizing the Jianghan Plain’s ecological network. This study provides a novel framework and theoretical support for regional habitat, biodiversity conservation, and sustainable development.