Conjugated linoleic acid (CLA) enriched triacylglycerol (TAG) of 90 wt% was successfully synthesized in 10 h by direct esterification of glycerol and CLA using an immobilized lipase from Candida antarctica under vacuum. The best operating conditions for the synthesis of TAG were investigated according to the three parameters of temperature, enzyme loading, and vacuum. The synthesis of TAG increased with increasing temperature but it did not significantly change above 60°C (p > 0.05). The increase of enzyme loading lead to enhanced conversion of TAG, but enzyme loading of more than 10% (based on the total weight of the substrate) was not effective. Moreover, when vacuum increased, the conversion of TAG increased, but the conversion rate decreased at too high vacuum level. The best combination of temperature, enzyme loading, and vacuum level were 60°C, 10% of the total weight of the substrate, and 0.4 kPa, respectively. During the initial 6 h of reaction, Candida antarctica lipase had more selectivity for 10t,12c‐CLA than 9c,11t‐CLA onto the glycerol backbone, and preference for incorporation at the sn‐1,3 positions of glycerol rather than at the sn‐2 position.