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Simple Summary: The health of Amphioctopus fangsiao, a species used in aquaculture or fish farming, can be greatly affected by infections. To understand how their immune system responds, we looked at the changes in their immune cells when exposed to substances that mimic these infections. By studying these responses, we were able to identify key parts of their immune system that change when faced with infection-causing threats. We also discovered important genes, including PKMYT1 (protein kinase, membrane associated tyrosine/threonine 1) and NAMPT (nicotinamide phosphoribosyltransferase), that play a crucial role in this response. Our study gives us a deeper understanding of the immune system of the Amphioctopus fangsiao. The primary influencer of aquaculture quality in Amphioctopus fangsiao is pathogen infection. Both lipopolysaccharides (LPS) and polyinosinic:polycytidylic acid (Poly I:C) are recognized by the pattern recognition receptor (PRR) within immune cells, a system that frequently serves to emulate pathogen invasion. Hemolymph, which functions as a transport mechanism for immune cells, offers vital transcriptome information when A. fangsiao is exposed to pathogens, thereby contributing to our comprehension of the species' immune biological mechanisms. In this study, we conducted analyses of transcript profiles under the influence of LPS and Poly I:C within a 24 h period. Concurrently, we developed a Weighted Gene Co-expression Network Analysis (WGCNA) to identify key modules and genes. Further, we carried out Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses to investigate the primary modular functions. Co-expression network analyses unveiled a series of immune response processes following pathogen stress, identifying several key modules and hub genes, including PKMYT1 and NAMPT. The invaluable genetic resources provided by our results aid our understanding of the immune response in A. fangsiao hemolymph and will further our exploration of the molecular mechanisms of pathogen infection in mollusks. [ABSTRACT FROM AUTHOR]