Utilization of algae oil and glycerol for preparation of value-added products is vital for sustainable development of related industries. In this study, we aimed to prepare highly pure diacylglycerols (DAG) rich in docosapentaenoic acid (DPA). First, content of DPA in form of triacylglycerols (TAG) increased from 16.4% to 28.1% after low-temperature crystallization of Schizochytrium sp. oil at -80 degrees C for 6 h. Subsequently, DPA-enriched DAG was prepared by the enzymatic glycerolysis of the enriched oil. Under the optimum conditions, there was 48.4% DAG produced in the crude mixture. To remove polar impurities from the crude product, a novel two-step purification was developed and the final product consisted of 75.1% DAG and 24.9% TAG with a low peroxide value. The current method for the synthesis of DAG rich DPA is effective and relatively mild and the successful preparation of value-added product will reduce production costs for algae and biodiesel industries.