Arachidonoyl ethanolamide (AEA) is a bioactive lipid that occurs naturally in animal and plant tissues. In this study, arachidonic acid (ARA) was enriched first from a type of microbial oil by a two-step purification, and the purified ARA concentrate was then used for the enzymatic preparation of AEA by amidation between ARA and ethanolamine. The purity of initial ARA increased from 44.4% to 85.1% after urea complexation and acetonitrile extraction. The purified ARA was subsequently used for lipase-catalyzed synthesis of AEA. The results showed that under optimized conditions (5 mmol purified ARA product, 5.5 mmol ethanolamine, 3 mL hexane, 10% Lipozyme 435, 70 degrees C for 2 h), fatty acid ethanolamide was obtained with 95.6% yield. The current method for making AEA by the enzymatic amidation of ethanolamine with ARA from fungal oil is effective, practical and economically feasible.