Abstract: China is advocating ecological civilization construction nowadays. Further researches on the relation between ecosystem service and humanity well-being are full of theoretical and practical significance. Combining related researches, this paper defined the concept and connotation of ecological well-being based on ecosystem service theory. Referencing theory of national economic accounting and relative researches, the evaluation indicators of ecological well-being supply and consumption were established. The quantitative characterization and evaluation method of red line of regional ecological well-being was proposed on the basis of location quotient. Then the evaluation of ecological well-being in mainland China in 2012 was set as an example for empirical research. The results showed that the net product values of 6 ecosystems, includingcultivated land, forest land, grassland, wetland, water area and unused land, were respectively 1481.925, 8194.806, 4176.277, 4245.760, 3177.084 and 133.762 billion CNY. Spatial heterogeneity of ecosystem net product in different provinces was distinct. Ecological well-being per capita of forest land, grassland, wetland, cultivated land and unused land in eastern and middle provinces were under the red line and less than the national average. The spatial distribution of 9 kinds of ecological well-being per capita split at Hu’s line with high value in northwest and low value in southeast, and was aggravated by differences in density of population and land resources gift.