In order to cope with the major challenges of economic transformation and mass entrepreneurship, the entrepreneurial ecosystem development of innovation space (IS) has become the top priority of our government to promote mass entrepreneurship and innovation, and government regulation is a necessary means to ensure the healthy development of entrepreneurial ecosystem of IS. The impacts of government regulation and initial willingness of the three parties on the entrepreneurial cooperation between IS and EV is analyzed by MATLAB simulation software and evolutionary game theory. The research finds that: (1) Compared with the mode of government’s direct subsidies to entrepreneurial ventures (EV), the government’s subsidies to IS is more conducive to the evolution of IS to provide quality entrepreneurship service and EV towards acceptance incubation. (2) In the early stage of the development of IS, the government’s behavior strategy evolved towards positive regulation; when entrepreneurial ecosystem of IS matured, the government’s behavior strategy turns negative. (3) Compared with government regulation, the initial willingness level of the government, IS and EV has more significant impact on the evolution of the behavior strategies of each subject. When the initial willingness of the government regulates actively, the willingness of IS providing quality entrepreneurship service and EV accepting incubation is low, the impact of government regulation on the evolution of IS and entrepreneurial venture’s behavior is negligible. But, when the initial willingness of each subject is high, the lower level of government regulation can achieve higher expected goals.