To solve the stability and oxidation issues of alpha-linolenic acid (ALA), this study focused on developing ALA nanoemulsion system (ALA-NE, oil-in-water) and evaluating the effect of environmental conditions on physical stability and the effect of antioxidants on oxidative stability. The physicochemical properties of nanoemulsions were measured at different conditions, including particle diameter, zeta potential, retention rate and peroxidation value (POV). The particle diameter increased significantly and the retention rate decreased after 25 days storage under the conditions of high temperature and metal ions. However, the influence of ionic strength, pH and light was insignificant. As an antioxidant, Vitamin E was more effective at retarding lipid oxidation of nanoemulsions than that of vitamin C. These results provided reference information in preparing effective and stable ALA-NE systems and enlarging the application fields.