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Environmental and physiological fluctuations in the live oyster cold chain can result in reduced survival and quality. In this study, a flexible wireless sensor network (F-WSN) monitoring system combined with knowledge engineering was designed and developed to monitor environmental information and physiological fluctuations in the live oyster cold chain. Based on the Hazard Analysis and Critical Control Point (HACCP) plan to identify the critical control points (CCPs) in the live oyster cold chain, the F-WSN was utilized to conduct tracking and collection experiments in real scenarios from Yantai, Shandong Province, to Beijing. The knowledge model for shelf-life and quality prediction based on environmental information and physiological fluctuations was established, and the prediction accuracies of TVB-N, TVC, and pH were 96%, 85%, and 97%, respectively, and the prediction accuracy of viability was 96%. Relevant managers, workers, and experts were invited to participate in the efficiency and applicability assessment of the established system. The results indicated that combining F-WSN monitoring with knowledge-based HACCP modeling is an effective approach to improving the transparency of cold chain management, reducing quality and safety risks in the oyster industry, and promoting the sharing and reuse of HACCP knowledge in the oyster cold chain. [ABSTRACT FROM AUTHOR]