Although the methanolysis of microalgae oil for biodiesel preparation was studied previously, the ethanolysis for the combined production of fatty acid ethyl esters (FAEEs, also can be used as biodiesel) and the ethyl esters of PUFAs (an important health-care product) rarely reported. In this work, a novel two-step process, hydrolysis of microalgae oil followed by lipase-mediated ethanolysis, was developed for the synthesis of FAEEs and the ethyl ester of PUFAs. The hydrolysis was firstly performed and the generated free fatty acids was collected and further used for FAEEs synthesis catalyzed by lipase. For lipase-catalyzed ethanolysis, the adding strategy of methanol, molecular sieve dosage, lipase dosage and temperature had significant influence on the FAEE yield. Under the optimized conditions, a FAEE yield of 97% could be obtained. Novozym435 was found to be equally effective for catalyzing the ethanolysis of conventional free fatty acids and PUFAs. The combined effects of multiple factors on the ethanolysis was further studied using Response Surface Methodology (RSM). Validation statistical analysis suggested that the negative correlation existed between FAEE yield and the water content. This work provided a novel strategy to produce FAEEs and the ethyl esters of PUFA effectively with microalgae oil as the substrate. (C) 2019 Elsevier Ltd. All rights reserved.