Innovation is the first driving force of national development, industrial agglomeration is the key factor to promote the development of regional innovation. Based on the whole industry data of 108 prefecture level cities in the Yangtze River Economic Belt from 2009 to 2018, this paper uses data envelopment analysis (DEA) and other methods to reveal the spatial-temporal evolution characteristics of regional innovation efficiency. Secondly, it establishes a spatial econometric model to analyze the spatial spillover effect of industrial specialization and diversified agglomeration on regional innovation efficiency. The results show that: (1) on the whole, the regional innovation efficiency of the Yangtze River economic belt first rises and then declines, with the characteristics of stages; (2) industrial specialization and diversified agglomeration have a significant positive impact on the regional innovation efficiency; (3) from the perspective of spillover effect, diversified agglomeration has a stronger role in promoting the regional innovation efficiency of the adjacent areas. Therefore, it is recommended that downstream areas actively take advantage of the balance of development between regions, adopt a mixed mode of coexistence of multiple industries, and strive to build a modern metropolitan area; central cities in the midstream and upstream areas should focus on the diversified cluster of industries, and small cities should fully Give full play to its own resource advantages and enhance the level of industrial specialization and agglomeration.