The development of the fresh-food e-commerce has led scholars to pay more attention to research on the agricultural product supply chain. This paper analyses the operation mode of the new O2O retail fresh supply chain by constructing the Stackelberg game model, takes the freshness and freshness-keeping efforts of fresh agricultural products as the main considerations, and analyses and compares the overall income of the supply chain under different decision-making modes. The results of the study show that: (1) In a centralized decision-making model, collaboration between suppliers and retailers online and offline can increase their respective revenues, and overall supply chain profits increase as the proportion of collaboration increases; (2) compared to decentralized decision-making, revenue-sharing contracts can increase the overall profitability of supply chain members and the supply chain when the revenue sharing factor is relatively high in the case of online and offline channel collaboration; (3) finally, the impact of revenue-sharing contracts on supply chain profitability is discussed through numerical analysis.