Innovation is the first driving force, and it is necessary to build a national innovation system with regional coordination to drive development through innovation. Existing research shows that the implementation of the policy of building national innovation cities has effectively improved the innovation level of the pilot cities and driven the innovation development of other cities, but whether it has promoted regional coordinated innovation needs further research and lacks empirical evidence. This paper argues that the creation of national innovative cities will improve the innovation environment, gather innovation elements and enhance the innovation capacity of pilot cities, thus promoting the increase of innovation and entrepreneurship activities and accelerating the development of high-tech industries. The resulting growth in cooperative innovation demand and improvement of the cooperative innovation environment will further promote innovation cooperation between pilot cities and other cities, thus benefiting the development of regional collaborative innovation. The stronger the cooperative innovation capacity and the better the cooperative innovation environment of the pilot cities, the stronger the regional collaborative innovation promotion effect. This paper took the construction of national innovative cities as a quasi-natural experiment, used the data of 276 prefecture-level cities from 2003 to 2019, used the number of per capita cooperative patents to depict the cooperative innovation level between cities, and then used the double difference method to analyze the impact of pilot policy on the cooperative innovation of cities. The results found that the implementation of the pilot policy for the construction of national innovative cities has a significant positive effect on the cooperation and innovation between the pilot cities and other cities. This conclusion still holds after a series of robustness tests such as removing provincial capital city samples, PSM-DID test, pilot time lag treatment, controlling the influence of other pilot policies, and placebo test, indicating that the pilot policy effectively promotes regional collaborative innovation; the implementation of the pilot policy significantly promotes the innovation and entrepreneurship activities and the development of high-tech industries in the pilot cities, indicating that the creation of national innovative cities can promote regional collaborative innovation by accelerating the innovation development and industrial upgrading of the pilot cities; the regional collaborative innovation promotion effect of the pilot policy is more significant in the eastern region than in the central and western regions, indicating that improving the ability of cooperation and innovation and improving the environment of cooperation and innovation are conducive to the full play of the policy effect. Compared to existing literature, this paper expands the policy effect analysis of the national innovation city construction pilot from the perspective of regional collaborative innovation and provides empirical evidence for the promotion of innovation city construction on regional collaborative innovation. This paper analyzes the mechanism of innovation and entrepreneurship and the development of high-tech industry and studies regional heterogeneity, which is conducive to the improvement of pilot policies and the promotion of regional collaborative innovation. The research of this paper shows that the creation of national innovative cities has promoted regional collaborative innovation while accelerating innovation and high-tech industry development in the pilot cities. Therefore, it is necessary to continue to vigorously promote and extend the national innovation city construction pilot, build a reasonable layout of innovative city networks, strengthen innovation cooperation and innovation drive, and promote the formation of a high-level national innovation system with regional collaboration.