In the present study, high-purity linolenic acid products were obtained by two-pass chromatography of linseed oil with urea-silica gel and cuprous chloride-silica gel. The results showed that linseed oil contained only oleic acid, linoleic acid and linolenic acid after urea-silica chromatography. A linolenic acid product with a purity higher than 90% was obtained after cuprous chloride-silica chromatography. Methyl linolenic acid was used as the standard for the external standard quantitative method. The total linolenic acid of the sample showed good linearity in the range of 0.045~0.677 mg/mL. The regression equation was Y=782 59x-686 8(R2=0.999 9). This research will provide relevant theoretical basis and new ideas for the purification and industrial production of linolenic acid.