[Objective] An assessment framework for ecological security evaluation of natural resources was constructed to further analyze its evolution law in response to urbanization, in order to provie references for regional territorial space optimization and ecological civilization construction. [Methods] Based on the correlation between ecosystem health and ecosystem services, an ecological security framework based on “contribution, vigor, organization and resilience” was constructed. By using the InVEST model, landscape indices, and the Delphi method to evaluate the ecological security of Wuhan City in 2000 and 2021, the temporal and spatial evolution of ecological security was also analyzed. [Results] ① The ecological security index of Wuhan City was 0.394 4 in 2000 and 0.374 5 in 2021. Ecological security level showed a spatial distribution pattern of “low in the middle and high in the surrounding area”. The ecological security level of the Huangpi District was the highest. ② From 2000 to 2021, the overall ecological security of Wuhan City showed a downward trend. The declining areas were mainly located in a ring along the edge of the central urban area. The distribution of the “low security” area tended to gradually expand from the central core area. The transition of areas from “medium ecological safety” to “low ecological safety” and from “high ecological security” to “medium ecological safety” was widespread. [Conclusion] Urban expansion has resulted in declining ecological security of natural resources. Therefore, territorial space planning should be further strengthened, including punishments for illegal occupation of ecological land and strengthening the protection of ecological space.