Polyunsaturated fatty acids (PUFAs) have various physiological functions. Of these, ethyl eicosapentaenoate is industrially purified and used as a medicine. Other PUFAs, such as docosahexaenoic acid (DHA), gamma-linolenic acid (GLA), and arachidonic acid (AA), are also expected to be used as pharmaceutical agents; however, their industrial purification processes have not been established. Because PUFAs are highly unstable against heat and oxidation, we attempted to purify them by taking advantage of their enzymatic reactions. When free fatty acids (FFAs) originating from PUFA-containing oil were selectively esterified with lauryl alcohol (LauOH) using a lipase acting on a desired PUFA very weakly, the PUFA was efficiently enriched in the FFA fraction. In addition, when selective alcoholysis of ethyl esters originating from PUFA-containing oil with LauOH was carried out, the PUFA ethyl ester (EtPUFA) was enriched to a desired purity in the unreacted ethyl ester fraction. These reaction mixtures contain LauOH, PUFA (EtPUFA), and lauryl esters, and their molecular weights are different from one another. Hence, PUFA or EtPUFA can be easily separated by conventional distillation. Selective esterification increased the purity of DHA, GLA, and n-6 PUFAs rich in AA to 91, 98, and 96 wt%, respectively. Selective alcoholysis was also effective for increasing the purity of ethyl docosahexaenoate to 90 wt%.