Provided is a preparation process of diglycerides which comprises reacting a fatty acid or lower alkyl ester thereof with glycerin in the presence of an immobilized partial glyceride lipase while removing water, which is generated upon reaction, out of the system. The reaction is preferably terminated when the acid value (AV) of the reaction mixture satisfies the following range: 50R-60>AV>70R-150 (with the proviso that AV>0). The present process makes it possible to efficiently prepare a high purity of diglycerides without impairing lipase activity even under reduced pressure. The present process is effective without being influenced by variations in reaction conditions such as fatty acid/glycerin ratio, reaction temperature or enzyme concentration. Particularly, even when the fatty acid/glycerin ratio is high, a high purity of diglycerides can be produced at a high reaction yield.