Abstract: The online and offline O2O model has become an important means for the transformation and upgrading of the traditional retail industry. With the continuous deepening of the Internet and big data technologies, the boundaries of enterprises are becoming blurred, and information islands continue to merge. Enterprises have begun to move from one-way chain development to multilateral network cooperation, and gradually move towards the evolution and upgrade of the overall business ecosystem, symbiosis and co-creation. The co-acting cooperative thinking gradually replaces the traditional zero-sum game competitive thinking and becomes a new choice for companies to collaborate with the overall ecosystem to achieve value creation and growth.From the perspective of business ecosystem evolution, discuss the realization mechanism of the co-creation of multiple values in the retail industry under the background of the O2O business ecosystem, and take Yonghui Supermarket as an example to elaborate and analyze, pointing out: the four subsystems of core, support, competition and environment are the basis for the retail industry to realize multi-value co-creation under the O2O business ecosystem. Among them, the core system is the direct carrier of value creation, and its participants are the main body of value creation. The diversified interactions and evolutionary symbiosis constitute the realization path of the overall ecological diversified value creation, and the common sharing, inclusiveness and co-integration of mutual data provide it with evolutionary power.