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Abstract: With the expansion of human activities, human-dominated land cover conversion has become the most prominent cause of habitat fragmentation. Urbanization is currently one of the most significant factors driving land conversion and causing habitat fragmentation. Habitat isolation, as one major component of habitat fragmentation, is a dynamic process and complicated to evaluate and quantify. This paper intends to investigate habitat isolation due to rapid urbanization. Two new metrics, Urbanization Isolation Effect (UIE) and Habitat Isolation Degree (HID), are proposed to incorporate urban sprawl and population increase into the quantification of habitat isolation, and demonstrate its spatio-temporal variation. The Shenzhen River catchment, a cross-boundary region shared by Hong Kong and Shenzhen SEZ in China, has been used as a case study to demonstrate the effectiveness of these proposed metrics. The results show that (1) extensive land conversion to urban utility has occurred since 1988, especially on the Shenzhen side of the catchment; (2) the metrics of UIE and HID exhibited remarkable spatial and temporal variations in the whole catchment and also displayed a significant discrepancy between Hong Kong and Shenzhen; and (3) urban sprawl on the Shenzhen side exerted trans-boundary influences on habitats of the Hong Kong side. In summary, the two proposed metrics are proved to be effective in demonstrating the spatio-temporal variation of habitat isolation and its causes, as well as identifying the extent and intensity of the urbanization isolation effect. These metrics may be useful for regional planning and natural landscape conservation. [Copyright &y& Elsevier]