Normalized resistance interpretation (NRI) analysis for tetracycline was applied to generate information on the epidemiological cut-off value for Vibrio ichthyoenteri isolated from diseased olive flounder (Paralichthys olivaceus) larvae. Thus, 42 strains of V. ichthyoenteri were used to determine minimum inhibitory concentration (MIC) values of tetracycline using Etest. Also, 11 tetracycline resistance related genes were investigated by PCR method. Most tetracycline-resistant strains harbored both tetB and tetM with a few ceptions. NRI-derived mean and 2 SD above the mean of theoretical normal distributions of susceptible isolates were 0.33 mg/L and 1.66 mg/L, respectively. The epidemiological cut-off value for V. ichthyoenteri from the calculations could be set to S ≤ 2 mg/L. Of the 42 strains, 15 were classified as non-wild type (NWT), and MIC values of the NWT strains vary regardless of tetB and tetM detection, suggesting that there may be other mechanisms involved in tetracycline resistance in this Vibrio species.