Abstract 2-Arachidonoyl glycerol (2-AG) is an endogenous agonist for cannabinoid receptors and has exhibited various biological activities. In this study, we reported an improved method for the synthesis of 2-AG by enzymatic ethanolysis of arachidonic acid-rich oil. The effects of solvent type, addition amounts of solvent and lipase, and reaction time on the content of 2-monoacylglycerols (2-MAGs) in the crude reaction mixture were investigated. Under the optimal conditions, 34.1% (area/area) 2-MAGs were produced in the crude mixture. 2-MAGs were obtained at 98–99% purity and 85.3% 2-MAGs yield (mol/mol) after solvent fractionation to fully remove impurities, including diacylglycerols (DAGs), triacylglycerols (TAGs) and fatty acid ethyl esters. Subsequently, pure 2-MAGs were further purified by crystallization in hexane to remove all saturated and partially unsaturated 2-MAGs to enrich 2-AG. After a two-step purification, 79.4% 2-AG was obtained at 42.9% 2-MAGs yield. Compared to previous methods for the synthesis of 2-AG, the method reported herein is easier to scale, greener, simpler and more economical.