The invention relates to a method for separation of alpha-linolenic acid from Chinese prickly ash seed oil and belongs to the field of food additives and their preparation methods. The method comprises the following steps of adding Chinese prickly ash seed oil into a NaOH-ethanol solution, carrying out heating reflux, carrying out shaking until the solution is transparent, carrying out cooling to a room temperature, adding petroleum ether into the cooled solution, removing an excess amount of ethanol, adding water into the solution, adjusting a pH value to 2-3, transferring the solution into a funnel, carrying out extraction, carrying out standing, washing an upper layer organic phase by water until the organic phase has a pH value of 7, evaporating the filtrate, recovering petroleum ether, collecting mixed aliphatic acid, mixing ethanol, urea and the mixed aliphatic acid to obtain a uniform clathrate compound, transferring the uniform clathrate compound into a funnel, adding petroleum ether into the clathrate compound, adjusting a pH value to 2-3, adding water into the clathrate compound, carrying out extraction, carrying out standing, separating an organic phase, carrying out water phase extraction by petroleum ether, merging the organic phases, washing the organic phases until pH is 7, carrying out dehydration drying on the petroleum ether layer, and evaporating the filtrate to completely remove petroleum ether so that alpha-linolenic acid is obtained. The method utilizes a urea clathration method, is simple, economic and efficient, and realizes alpha-linolenic acid relative concentration increasing from 28.41% to 61.92% by one urea clathration process.