The invention provides a method for separating all-trans fatty acid with antitumor activity from antarctic krill oil. According to the method, the antarctic krill oil is subjected to silica-gel chromatography coupling, high-performance liquid chromatography is prepared to carry out separation and purification, antitumor activity detection is carried out in an assisting manner so as to determine effective ingredients, molecular weights, structural compositions and configurations of the effective ingredients are determined separately through high-resolution electro-spraying ionization mass spectrum, nuclear magnetic resonance and a laser Raman scattered spectrum, and the effective ingredients are identified as all-trans EPA and all-trans DHA. Discovered by comparison of median lethal concentration of antarctic krill, commercially-available deep-sea fish oil and Amur sturgeon fish liver EPA and DHA to a variety of tumor cells, the all-trans EPA and all-trans DHA originated from the antarctic krill oil have a good tumor cell growth resisting action and are free of obvious inhibiting effect on normal control cells, thereby having a potential antitumor candidate drug development prospect.