In order to explore the urban ecotourism development level differences and influencing factors of each region of the Yangtze River Delta and propose corresponding improvement strategies, this paper constructed an evaluation index system of urban ecotourism development level based on the essence of urban ecotourism and used entropy weight TOPSIS method to measure urban ecotourism development level of 41 cities in the Yangtze River Delta from 2010 to 2019. Then, we also used the standard deviation ellipse and Markov chain to analyze the spatio-temporal evolution characteristics of the level of urban ecotourism development. The Geodetector was used to explore the influencing factors of the development of urban ecotourism, which aimed to find a differentiated approach to make targeted suggestions for the improvement of the level of urban ecotourism development. The results showed that: (1) The growth rate of each dimension of urban ecotourism in the Yangtze River Delta was different. The supply and security were the main factors affecting the comprehensive development level. The spatial pattern of each dimension was significantly different. (2) The comprehensive development level of urban ecotourism in the Yangtze River Delta showed a fluctuating upward trend, and the growth rate was slow. The spatial distribution of the development level of each city was unbalanced, and a stable distribution pattern of high in the southeast-low in the northwest has initially formed in space. (3) The center of gravity of the level of urban ecotourism development in the Yangtze River Delta presented a shift pattern of northwest-southeast-northwest, with the degree of agglomeration to the southeast gradually increasing. The development of urban ecotourism exhibited a slow and gradual evolutionary process, and the horizontal level shift only occurred between adjacent levels. (4) The development of urban ecotourism in the Yangtze River Delta was influenced by a variety of factors. The influence of the level of transportation development and urbanization was more significant, the influence of economic development level and resident income level was gradually increasing, and the promotion effect of the level of industrial structure and environmental protection emphasis was not significant. The interaction between any two factors was greater than the force of a single factor and it showed either a two-factor enhancement or a nonlinear enhancement. The results of the study can provide certain theoretical basis and scientific reference for the Yangtze River Delta to improve the level of urban ecotourism development and comprehensively promote the sustainable development of tourism.