1,3-Diacylglycerol is conducive to reducing the increase of body fat and slowing the raise of body weight. So, growing attention has been paid to the enzyme-mediated 1,3-diacylglycerol preparation due to the excellent selectivity and the environmental friendliness. In this work, we provided an alternative and sustainable process for 1,3-diacylglycerol synthesis catalyzed by immobilized lipase from Penicillium expansum (PEL) in which the esterification of oleic acid with glycerol for 1,3-diolein synthesis was employed as the model reaction. The results indicated that, under the optimum conditions (10.0 g of oleic acid, 1.09 g of glycerol, 80 U of immobilized PEL, and 6.4 g of 4 Å molecular sieves at 40 °C for 12 h), the diolein yield achieved to 82.4%, and the value of 1,3-diolien/diolein was 87.3%. Additionally, 92% original activity of the immobilized PEL was maintained after being used for 10 batches, highlighting the presumable cost-effectiveness of the enzyme. This study could be considered as an alternative and green strategy for preparing 1,3-diacylglycerol.