Entrepreneurship, as one of the core driving forces of economic vitality, continuously injects fresh blood and growth momentum into the high-quality economic development by providing employment, industrial upgrading and structural transformation. Meanwhile, standards, as the institutional cornerstone for the construction of a unified national market, provide an important guarantee for the prosperity and development of the urban entrepreneurial ecosystem. Since 2016, the State Council has successively approved five provinces including Zhejiang, Jiangsu, Shandong, Shanxi and Guangdong to launch national comprehensive reform pilot actions for standardization. Each region focused on areas such as technological innovation, public services and internationalization environment, and carried out comprehensive governance reforms in aspects such as standardization system construction, institutional mechanisms and supply capacity. Based on the theory of entrepreneurial ecosystem, the implementation of the national comprehensive reform pilot policies for standardization is regarded as a quasi-natural experiment. Using the panel data of 277 prefecture-level cities in China from 2007 to 2022, a multi-time-point difference-in-differences model was constructed to examine the empowerment effect of standardization governance on urban entrepreneurial vitality and the influencing mechanism. The research findings are as follows: (1) The standardized governance actions represented by Chinese Standardization Comprehensive Reform Pilot significantly enhanced the entrepreneurial activity of cities. This conclusion remained valid after a series of robustness tests such as parallel trend test, placebo test, substitution of dependent variables, and change of research methods; (2) The mechanism test indicates that technological innovation clustering and public service optimization played a mediating role in activating the entrepreneurial vitality of cities through standardized governance, while the enrichment of human resources and high-level opening-up positively regulated the incentive effect of standardized governance on the level of urban entrepreneurship; (3) The enhancement effect of the national comprehensive reform pilot policies on the entrepreneurial activity of cities varies due to different urban characteristics such as geographical location, education investment, industrial environment and marketization degree. Specifically, the entrepreneurial incentive effect of standardized governance is more obvious in northern cities, cities with greater education investment, better industrial environment and higher marketization degree. The research conclusions of this paper provide new ideas for activating the urban entrepreneurial ecosystem, explore the boundaries of standardized governance, and offer certain references for building a unified national market and promoting high-quality economic development. The possible contributions of this paper are as follows: First, by constructing a quasi-natural experiment based on the national comprehensive reform pilot of standardization, this paper for the first time evaluated and demonstrated the entrepreneurial incentive effect of standardized governance, not only expanding the influence boundary of standardized policies, but also enriching the entrepreneurial research system in the Chinese context from the institutional level. Second, based on the comprehensive application of the entrepreneurial ecosystem theory and Schumpeter's innovation theory, this paper deeply reveals the internal mechanism of standardized governance activating the urban entrepreneurial ecosystem from multiple aspects such as technological innovation, public services, human resources endowment and opening-up, enriching the research content of government policies' impact on urban entrepreneurship, and also based on geographical location and urban characteristics, carefully examines the heterogeneity facts of policy effect. Third, starting from stimulating the entrepreneurial vitality of cities, this paper connects the formulation of government policies and the commercial behavior of market entities from the perspective of standardized governance, providing a theoretical logic and empirical evidence for the combination of an active government and an effective market to jointly exert efforts and promote innovation development.