To investigate the relationship between metabolic changes in 1H-nuclear magnetic resonance (NMR) spectra and fish vaccination, serum was collected from olive flounders treated with a formalin-killed Edwardsiella tarda vaccine and use for H1-NMR metabolite profiling. Principal component analysis and partial least squares were applied to the 1H-NMR profile to reduce its complexity and establish class-related clusters. Relative lipid regions were distinguished in vaccinated and non-vaccinated serum. Then, the lipids were extracted from the serum and analyzed. Triolein was identified.