Diacylglycerol (DAG) is functional oil with many beneficial effects. However, the current approaches for DAG preparation face the problems of the low utilization rate of substrates and producing large amounts of by-product. Here, we report a novel approach for simultaneous preparation of edible quality medium and high purity DAG by combining enzymatic partial hydrolysis and a novel partial glyceride lipase (PGL)-catalyzed esterification. Firstly, four lipases were screened for their capability in DAG preparation by partial hydrolysis and Lipase DF “Amano” 15 was the best. Under the optimal conditions, scale-up reaction was performed and residues containing 55.2% DAG were obtained after molecular distillation. Subsequently, the collected distillates were used as substrate for DAG synthesis by a novel PGL-catalyzed esterification and PGL AOL-V269D was selected. Under the optimized conditions, scale-up reaction was performed and residues containing 99.3% DAG were obtained after molecular distillation. The collected distillates were recycled for further DAG synthesis. Finally, the obtained medium and high purity DAG were characterized, lacking glycidyl esters and 3-chloro-1,2-propanediol esters, and with low acid and peroxide value reaching the edible quality. Overall, the proposed novel procedure enabled the substrates to be used extensively without by-product production.