The technology of enriching palmitoleic acid from fish oil fatty acid ethyl ester by urea complexation and molecular distillation was studied. The conditions of urea complexation were reaction temperature： 75 ℃， mfishoil ∶ murea ∶ methanol = 1 ∶ 2 ∶ 4. The effects of scrape speed， feed rate and distillation temperature on palmitoleic acid content were investigated through the single factor experiment. On the basis of single factor experiment， the molecular distillationwas optimized by response surface experiments with Box-Behnken design. The final experiment showed that the optimum acetylation conditions were scrape speed 140 r/min， feed speed 1 mL/min， distillation temperature 65 ℃. Under these conditions， the content of palmitoleic acid in the product increased from 8.41% to 32.29%， and the recovery rate was 60.5%.