Platycladus orientalis L. (Cupressaceae) has a lifespan of thousands of years. Ancient trees have very high scientific, economic and cultural values. The senescence of ancient trees is a new research area but is poorly understood. Leaves are the primary and the most sensitive organ of a tree. To understand leaf structural response to tree senescence in ancient trees, experiments investigating the morphology, anatomy and ultrastructure were conducted with one-year leaves of ancient P. orientalis (ancient tree >2,000 years) at three different tree senescent levels (healthy, sub-healthy and senescent) at the world’s largest planted pure forest in the Mausoleum of Yellow Emperor, Shaanxi Province, China. Observations showed that leaf structure significantly changed with the senescence of trees. The chloroplast, mitochondria, vacuole and cell wall of mesophyll cells were the most significant markers of cellular ultrastructure during tree senescence. Leaf ultrastructure clearly reflected the senescence degree of ancient trees, confirming the visual evaluation from above-ground parts of trees. Understanding the relationships between leaf structure and tree senescence can support decision makers in planning the protection of ancient trees more promptly and effectively by adopting the timely rejuvenation techniques before the whole tree irreversibly recesses.