The concept of the road-region ecosystem is described and its environmental features analyzed. In addition, the stability of the road-region ecosystem is summarized. A scientific and rational evaluation of road-region ecosystem stability is proposed to properly investigate the relationship between highway construction and protection of the ecological environment. This paper investigated a method for determining an index system to be used to evaluate road-region ecosystem stability. It put forward an index system for assessing road-region ecosystem stability as a reference. On the basis of detailed analysis of the multidimensional space of the road-region ecosystem, a new method and calculation formula for multi-objective comprehensive evaluation of road-region ecosystem stability are presented. Computation result is fit with the actual situation. The result indicates that the evaluation index system and the method are feasible.