How to effectively regulate the spread of Internet public opinion from multisubject is the focus that governments around the world need to face. From the cross perspective of multilayer network and multisubject association, this paper studies the diffusion mechanism of Internet public opinion, constructs the multilayer network of Internet public opinion and its diffusion model, and then systematically analyzes the evolution characteristics of Internet public opinion diffusion. The main conclusions of the study are as follows: it is easier to trigger the spread of Internet public opinion by establishing associations among netizens in a preferred way. When the probability of random connection in different layers increases synchronously, the diffusion intensity of Internet public opinion shows a gradual downward trend. The intensity of government intervention has the greatest impact on the diffusion intensity of Internet public opinion. When the factors of netizens, Internet platform, and government interact, it can effectively reduce the intensity and scope of Internet public opinion diffusion. Once the media participate, it will improve the complexity of Internet public opinion diffusion. The government’s intervention in Internet public opinion is a “double-edged sword,” which needs to set the intervention intensity according to different subjects and their factor characteristics. This study can provide a theoretical reference for formulating strategies to adjust Internet public opinion diffusion from the cross perspective of multilayer network and multisubject association.