Aiming at safety regulation in the operation of major infrastructure projects (MIPs) to prevent potential risk loss and adverse social impacts, this research presents a novel model integrating evolutionary game and system dynamics (SD) for optimizing safety regulation strategies with different stakeholders involving the operating company (OC), government section (GS), and public under the bounded rationality, where the evolutionary game theory is applied to describe the interactions among stakeholders in the safety regulation of MIPs followed by simulating through adopting the SD to analyze the effects of different strategies on equilibrium solutions and the stability of game equilibrium. In view of the simulation results based on five scenarios, the dynamic penalty-incentive scenario not only effectively restrains the fluctuations of the strategy selection, but also provides an ideal evolutionary stable strategy, in which the OC could nearly choose to comply with the regulations, while the public could nearly choose to supervise the OC as their optimal strategy to prevent risks. All results indicate that the application of the evolutionary game with the SD model is an effective way to analyze the effects of different strategies and provide effective solutions to study complex multi-player game problems. Overall, this research contributes to developing an evolutionary game with the SD model for the safety regulation of MIPs, which can serve as a platform to identify reasonable regulatory strategies with great practical application.