A production method of highly unsaturated fatty acids with a high purity/high yield that compensates for shortcomings of conventional techniques is provided.A purification method of highly unsaturated fatty acids and/or derivatives thereof comprising (a) contacting and stirring first raw materials comprising the substances with a first silver salt aqueous solution to collect a first oil layer and a first aqueous layer; (b) separating the first aqueous layer into a second silver salt aqueous solution and the substances; and (c) contacting and stirring the first oil layer with the second silver salt aqueous solution for separation into an oil layer and an aqueous layer to obtain a second aqueous layer comprising the substances.