Urban ecosystem health assessments can be applied extensively in urban management to evaluate the status quo of the urban ecosystem, identify the limiting factors, identify key problems, optimize the scheme and guide ecological regulation. Regarding the multi-layer roles of urban ecosystems, urban ecosystem health should be assessed at different scales with each assessment providing a specific reference to urban management from its own viewpoint. Therefore, a novel framework of multi-scale urban ecosystem health assessment is established on global, national, regional and local scales. A demonstration of the framework is shown by using a case study in Guangzhou City, China, where urban ecosystem health assessment is conducted in the order of global, national, regional, and local scales, from macro to micro, and rough to detailed analysis. The new multi-scale framework can be utilized to generate a more comprehensive understanding of urban ecosystem health, more accurate orientation of urban development, and more feasible regulation and management programs when compared with the traditional urban ecosystem health assessment focusing at the local scale.