2-Arachidonylglycerol (2-AG) has a wide range of biological and pharmaceutical applications. A three-step method was developed to synthesize 2-AG. Firstly, polyunsaturated fatty acid (PUFA) was enriched from saponified single cell oil by means of low temperature solvent crystallization, which gave a PUFA-rich fatty acid fraction with 74.71 wt% arachidonic acid and a corresponding yield of 97.1%. Secondly, triacylglycerol was obtained via lipase-catalyzed esterification of PUFA-rich fatty acid with glycerol. The TAG contents in the glyceride fraction reached 96 wt% with a degree of esterification of 94% after 6 h. Finally, the ethanolysis of TAG catalyzed by Novozym 435 resulted in regioisomerically pure (> 98 mol%) 2-monoacylglycerol containing 96.5 mol% PUFA. This final product contained 72 mol% 2-AG with a yield of 25.76% (the theoretical yield is 33.3%). When tri-arachidonylglycerol (purity 96 mol%) was used as the substrate in the third step, 2-AG with purity and yield of 97.1 mol% and 27.3%, respectively, was obtained. Complete assignment of the H-1 and C-13 NMR chemical shifts of 2-AG was carried out by means of two-dimensional NMR spectroscopy.