Megacities around the world are increasingly confronted with conservation and restoration bottlenecks due to the competing demands of urban expansion and environmental conservation. This study investigates conservation prioritization strategies for balancing biodiversity protection, ecosystem service (ES) supply and landscape connectivity in rapidly urbanizing Beijing. By employing spatially explicit modelling and prioritization scenario techniques, we identify spatially heterogeneous priority zones. We demonstrate that high-value areas for ES supply, particularly carbon storage and water regulation, concentrate primarily in Beijing’s north-western mountainous regions, covering c. 62% of the city’s area. Conversely, critical habitats for threatened species and key connectivity corridors are dispersed, with 22.89% of critical habitats located within urban built-up areas. Gap analysis reveals limited alignment between Beijing’s current ecological security patterns, with only 9.6% coverage of the identified top 10% conservation priority zones, especially within the metropolitan core. The study underscores significant trade-offs among different ecological objectives and multi-criteria conservation strategies. We propose an optimized conservation framework based on zonation analysis to guide targeted landscape planning decisions. This approach provides actionable insights for urban policymakers to achieve comprehensive sustainability, emphasizing the importance of protecting critical ecological areas in both urban and rural landscapes amid ongoing urban expansion.