As an important ecological corridor in China, the Yellow River Basin (YRB) plays an important role in maintaining China's ecological security. Understanding the spatial interactions between urbanization and ecosystem services is the basis for sustainable regional development. Based on the characteristics of urbanization development and the spatial distribution pattern of ecosystem services in the YRB from 2000 to 2018, the spatial and temporal interaction relationship and evolutionary characteristics of urbanization and ecosystem services in the YRB were further revealed using Pearson correlation coefficient and bivariate spatial autocorrelation. The results showed that:(1) The urbanization of the YRB had a significant spatial heterogeneity, and the comprehensive urbanization index increased from 2000 to 2018, with a growth rate of 102.60%, among which the development of economic urbanization was generally faster than that of land urbanization and population urbanization. (2) Food production, carbon sequestration services and soil conservation in the YRB increased by 65.32%, 59.47% and 46.27%, respectively, while water yield decreased by 21.15%. (3)The relationship between urbanization and ecosystem services in the YRB from 2000 to 2018 showed a weakening of the positive effect and an increase of the negative effect at both spatial and temporal scales, and the areas where the development of urbanization is not coordinated with ecosystem services are expanding.