High purity polyunsaturated fatty acids (> 95%) are essential for the synthesis of specialized pro-resolving lipid mediators (SPMs), such as protectins, resolvins, and maresins, which are used for clinical application. To date, high purity (> 95%) eicosapentaenoic acid (EPA; C20:5n3) and docosahexaenoic acid (DHA; C22:6n3) have been produced through various manufacturing steps using fish oil. In this study, we optimized preparative high performance liquid chromatography (HPLC) process to purify high-purity DHA ethyl ester (DHAee; > 98%) from oleaginous microalgae Shizochytrium sp. SH103 containing at least 34% DHA content. The purity and yield of DHA were determined by reverse phase chromatography with changing the mobile phase velocity, loading amount, and mobile phase composition. On a semi-preparative scale, optimal DHA separation in isocratic elution was obtained with a mobile phase velocity of 0.5 mL/min, a loading amount of 10 mg/mL, and mobile phase composition of methanol/water (96:4, v/v), wherein the purity of DHA was 98.5%. This separation was scaled up to a preparative column, resulting in 99.0% DHA fraction with a yield of 79.8%. This result suggests that a large amount of high purity DHA can be produced from microalgae when scaling up a preparative column to an industrial column.