Human activities during the process of urbanization continuously impact the structure and processes of ecosystems, even leading to irreversible losses in biodiversity. Investigating the mechanisms by which urbanization affects biodiversity can provide a reference for the formulation of biodiversity conservation strategies in China. Based on data regarding the level of urbanization and biodiversity, and guided by systematic conservation planning theory, this study uses methods such as Pearson correlation and random forest to explore the impact mechanisms of urbanization on biodiversity, delineating priority areas for biodiversity conservation in China. The results can offer decision support for adjusting China's biodiversity conservation strategy. The research findings are as follows: (1) High levels of urbanization are mainly located to the east of the "Hu Line," exhibiting a clear core-periphery characteristic. Over 30 years, the overall level of urbanization has increased by 100.00%. High biodiversity areas are primarily found in southern regions, with relatively lower biodiversity in eastern regions. Except for the Yangtze River Basin and the Inland River Basin, other regions have seen a significant decrease in overall biodiversity over the past 30 years; (2) There is a significant trade-off relationship between urbanization and biodiversity, and this trade-off is still intensifying. Among these, spatial urbanization has a particularly pronounced effect on biodiversity. (3) Biodiversity in the western watersheds of the "Hu Line" is more sensitive to population urbanization, while biodiversity in the eastern watersheds is more sensitive to economic and spatial urbanization; (4) The designated priority areas for biodiversity conservation are mainly located to the west of the "Hu Line," covering 28% of the national territory. The results reveal the regional differences in the impact of China's urbanization process on biodiversity, reflecting the necessity and urgency of demarcating priority areas for biodiversity conservation.