Abstract: The fatty acids of walnut oil were separated primarily by urea adduction method, then the linolenic acid of walnut oil was further purified by silver nitrate-silica gel column chromatography. In the silver silica gel column formed by 20 g silica gel and 2g Ag NO3, join in the ratio was 1∶15 with adsorbent after urea inclusion enrichment of fatty acids, 100 m L 8% acetone-petroleum, underd the elution flow rate of 0.6m L/min, the content of linoleic acid in walnut oil increased from the original 8.7% to 95.9%. This suggests that the silver nitrate-silica gel column chromatography is a kind of high efficient separation method of linolenic acid.