To identify the evolutionary dynamics of strategic decision-making among three parties-government, agriculture-related enterprises, and university-in the context of agricultural collaborative innovation integrating government, industry, and university, this study employed a tripartite evolutionary game model based on the Triple Helix Theory to investigate the impacts of collaboration costs, benefits, incentives, and breach penalties on the evolution of relationships among the three parties in agricultural government-industry-university collaborative innovation. The results indicate that: 1) Innovation costs and their allocation are key factors affecting collaborative stability. Agribusinesses show high sensitivity to these costs, whereas academic institutions are less responsive. Risk costs remain secondary unless the threshold is exceeded, as all parties prioritize deterministic costs and benefits. 2) The three parties differ in their demands for collaborative benefits. The government exerts macro-regulatory influence to sustain collaboration, agribusinesses focus on short-term economic benefits, while academic institutions emphasize non-economic factors. 3) Compared to the positive incentive coefficient, the increase in the frequency of collaborative innovation exerts a more significant promoting effect on synergy innovation. The government’s financial and policy incentives should be differentially designed according to the distinct response patterns of agribusinesses and academic institutions. 4) When penalty levels are low, system stability depends heavily on government oversight. Agribusinesses are particularly sensitive to penalties targeting themselves, while academic institutions are influenced by penalties applied to both sides. Furthermore, the effects of benefit loss and project progress display distinct phase-dependent characteristics. Taken together, stable government-industry-university collaborative innovation in agriculture requires a reasonable allocation of innovation costs, the reconciliation of parties’ benefit expectations, and differentiated incentives and constraints across actors and stages of collaboration.
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