In this study, we investigated the immunostimulatory and protective effects of CpG motif oligonucleotides (CpG-ODNs) against Edwardsiella tarda infection in olive flounder (Paralichthys olivaceus). Groups of fish injected with CpG-ODNs (1585, 1668, and 2007) or PBS (control) showed varying mortality rates in response to challenge with E. tarda. The survival rates of fish treated with CpG-ODN 1668 and 2007, which belonged to the same class type B, were 45% and 60%, respectively, with CpG-ODN 2007 showing the highest survival rate. Further analysis showed that the respiratory burst and bactericidal activities induced by CpG-ODN 2007 were higher than those in the control group (induced by non-CpG-ODNs) or in the group of fish induced by CpG-ODN 1585, which belonged to class type A. Additionally, the respiratory burst activity induced by CpG-ODN 2007 was higher than that induced by CpG-ODN 1668, despite similar bactericidal activity titers. In vivo experiments showed that CpG-ODN 2007 stimulation resulted in higher survival rates than CpG-ODN 1668 stimulation, possibly owing to differences in respiratory burst activity. In summary, we demonstrated that differences in CpG-motif or class type altered respiratory burst and bactericidal activities, resulting in differences in survival rates against E. tarda challenge in the olive flounder. Therefore, it is necessary to use CpG-ODNs optimized against E. tarda infection in olive flounder, because different CpG motifs belonging to the same class type have different effects.