1,3-diacylglycerol (DAG) is a natural lipid and known as functional oil. Here, we evaluated the potential of different natural deep eutectic solvents (NADESs) in synthesis of 1,3-DAG through Candida antarctica lipase B (CALB)-catalyzed esterification. Interestingly, we found that the 1,3-DAG content could be increased by 19.21 %, 13.10 %, and 8.74 % in three choline chloride (ChCl)-based NADESs, respectively, compared with that in solvent-free system. Confirmatory experiments demonstrated that NADESs could inhibit the acyl transfer of 1,3-DAG through modulating the water activity of the reaction system. Meanwhile, circular dichroism spectra and fluorescence spectroscopy results indicated that NADESs could not change the secondary structure of CALB, but could affect the tertiary conformation of CALB. Furthermore, molecular dynamics studies showed that the NADESs could induce a more compact tertiary conformation of CALB and enhance the sn-1,3 positional selectivity of CALB. Overall, the NADESs could induce a higher 1,3-DAG yield through inhibiting the acyl transfer of 1,3-DAG and enhancing the sn-1,3 positional selectivity of CALB. This study provides theoretical support for enzymatic production of structural lipids in NADESs.