In a new era of high-level opening up, transnational collaborative innovation has become an important way to foster growth drivers for emerging industries and achieve high-quality economic development. This paper breaks through the limitations of existing studies that focus on the impact of the “Belt and Road” Initiative on the innovation ability of a single enterprise, and switches the perspective to the win-win cooperation between Chinese enterprises and countries along the “Belt and Road”. Based on the data of Chinese A-share listed companies from 2010 to 2020, this paper takes the “Belt and Road” Initiative as a quasi-natural experiment, and uses the method of PSM-DID to investigate the impact of the “Belt and Road” Initiative on transnational collaborative innovation, and its mechanism and application situation. It is found that the “Belt and Road” Initiative effectively promotes the transnational collaborative innovation between Chinese enterprises and countries along the route through the two effects of “strengthening the flow and integration of factors” and “reducing the costs of transnational cooperation”. Heterogeneity analysis shows that when enterprises have strong absorptive capacity, belong to technology-intensive and capital-intensive industries, and have a high degree of cultural integration with countries along the route, the “Belt and Road” Initiative has a more significant effect on promoting transnational collaborative innovation of enterprises. Further research finds that the introduction of the “Belt and Road Special Plan for Scientific and Technological Cooperation” significantly strengthens the promotion effect of the “Belt and Road” Initiative on transnational collaborative innovation. This paper provides theoretical reference and practical evidence for the driving mechanism of transnational collaborative innovation between Chinese enterprises and countries along the “Belt and Road”, and also provides important enlightenment for micro-enterprises to realize transnational collaborative cooperation with the power of policies and empower their high-quality development.