Recently, studies have been carried out to utilize microalgae as a bio-transportation fuel due to environmental pollution problems such as global warming and problems caused by fossil fuels. In this study, the separation process of high-value product, DHA, was optimized for commercialization of microalgae transportation fuels. Transesterification reaction for oil conversion was optimized. In addition, urea complexation and the thin film evaporation method were used to separate DHA. Oil analysis was carried out quantitatively through gas chromatography. Conditions were optimized for each process. The content of DHA in the oil increased from 43% to 73%, meaningful results were obtained.