Grouper has become an essential mariculture species in China, while vibriosis caused by Vibrio harveyi significantly impacts its culture. Our previous study confirmed the V. harveyi formalin-killed cells vaccine (FKC) combined with chitosan oligosaccharide (FKC+COS) effectively prevents vibriosis. As an adjuvant, COS could significantly enhance FKC effectiveness against V. harveyi in grouper. In the present study, we performed transcriptome analysis of grouper spleens tissue 14 days post-immunization of PBS and FKC+COS, respectively. After assembly and annotation, 2,503 differentially expressed genes (DEGs) were obtained, including the upregulated 1,894 DEGs and downregulated 609 DEGs between the PBS group and FKC+COS group. To explore the relevance of DEGs in immunity, enrichment analysis in the KEGG database revealed that the main pathways of DEGs distribution associated with immunity were antigen processing and presentation, lysosome, the intestinal immune network for IgA production and FcγR-mediated phagocytosis. In conclusion, transcriptome analysis of spleens was performed to explore the potential mechanism of COS as an adjuvant enhancing the protection effectiveness of FKC against vibriosis in grouper.