Under the strategic background of new urbanization and "mass entrepreneurship and innovation", it is of great significance to analyze the internal laws of entrepreneurship at the city level for scientifically constructing an entrepreneurial support system and promoting the highquality urban development. Based on the theory of entrepreneurial ecosystem, this paper proposes an analytical framework of entrepreneurship, and empirically explores the impact of entrepreneurial ecosystem on entrepreneurship in the cities of Yangtze River Economic Belt, an important strategic region, by using urban panel data and Spatial Panel Dubin Model. The results show that: 1) The spatial distribution and spatial growth trend of entrepreneurship in the cities of Yangtze River Economic Belt show obvious spatial and temporal differentiation characteristics, and the overall trend of entrepreneurship concentration continues to increase. 2) The entrepreneurial ecosystem of the city has a differentiated impact on entrepreneurship in the Yangtze River Economic Belt. The influence of market environment, government size, innovation support, physical infrastructure is confirmed, but the impact of financial capital and human capital is not significant. 3) The spatial spillover effect of entrepreneurial ecosystem in neighboring cities cannot be ignored. The influence of market environment, government size and physical infrastructure is dominated by negative effects, while innovation capability is dominated by positive effects. 4) The study further proposes countermeasures and suggestions for improving the entrepreneurial support system from the aspects of the adjustment of the regional spatial structure of the Yangtze River Economic Belt, and the construction of urban entrepreneurial ecosystem [ABSTRACT FROM AUTHOR]