Abstract: Quadratic rotation-orthogonal combination design was applied to optimize the enrichment and purification techniques of α-linolenic acid in Eucommia ulmoides seed oil by the method of urea adduction fractionation. By taking α-linolenic acid purity as the investigation index, the effects of four process parameters named ratio of urea to fatty acid, ratio of 95% ethanol to fatty acid, inclusion temperature and inclusion time on the enrichment results were investigated and the two times multiple regression equation model of α-linolenic acid purity was established based on the single factor experiments. The results showed that the optimum levels of the four parameters were as follows:3:1, 9:1, -9. 0℃ and 17. 0h, respectively. Under the optimum enrichment conditions, the purity of α-linolenic acid was up to 82. 63%. Urea adduction fractionation was an effective method for the enrichment of α-linolenic acid in Eucommia ulmoides seed oil.