Uneven regional distribution of innovation resources due to administrative barriers and factor mobility constraints has seriously impeded cross-regional and cross-organizational joint innovation activities. Based on the panel data of 204 city pairs in 19 city clusters in China from 2011 to 2019, this paper establishes a multi-period double-difference model with the help of a quasi-natural experiment on the implementation of national city cluster policy to explore the impact of city cluster construction on the level of inter-city co-innovation and the way it works. The conclusions are as follows: the construction of city clusters effectively enhances the level of innovation cooperation among cities within city clusters, and this conclusion still holds after the instrumental variables are corrected for endogeneity and a series of robustness tests; geographic proximity, technological proximity and institutional proximity positively regulate the effect of the construction of city clusters, whereas organizational and cultural proximity are not major factors influencing the effect of the construction. Under the background of integrated development of city clusters, knowledge flows have directional characteristics, i.e., knowledge overflows from core cities to peripheral cities on the one hand, and on the other hand, flow barriers still exist between cities with different endowments. The conclusions of the study provide new ideas for the development and improvement of China’s future city clusters, optimization of economic structure and transformation of growth momentum.