Deep eutectic solvents (DESs) have numerous potential applications as cosolvents. In this study, use of DES as organic solvents for enzymatic biodiesel production from degumming palm oil (DPO) was investigated. Deep eutectic solvent was synthesized using choline chloride salt (ChCl) compounds with glycerol and 1,2-propanediol. Deep eutectic solvent was characterized by viscosity, density, pH and freezing values, which were tested for effectiveness by enzymatic reactions for the production of palm biodiesel with raw materials DPO. Deep eutectic solvent of ChCl and glycerol produced the highest biodiesel yield (98.98%); weight of DES was only 0.5 % of that of the oil. In addition, the use of DES maintained the activity and stability of novozym enzymes, which was assessed as the yield until the 6th usage, which was 95.07 % biodiesel yield compared with the yield without using DES. Hence, using DES, glycerol in enzymatic biodiesel production had high potentiality as an organic solvent for palm oil biodiesel production