Abstract: Response surface analysis method was applied to optimize the enrichment of α-Linolenic acid by freezing acetone method from Eucommia seed oil.According to the single factor experiments，optimizeing freezing temperature，V（acetone）/m（fattyacid）and freezing time by using Box-Behnken experiment design to optimize.And the content of α-Linolenic acid could be analyzed by GC-MS.The optimal conditions were determined as follows：freezing temperature -44 ℃，V（acetone）/m（fattyacid）4.2，freezing time 3.3 h.The actual α-Linolenic acid purity was 28.05 mg/mL and its content was 80.11%，compared to the theoretical value，the relative error of 0.43%.The content of α-Linolenic acid was improved from 60.14% to 80.11%.