In this study, the esterification reaction kinetics of biodiesel (fatty acid ethyl ester) prepared from waste oil (cooking oil waste and oleic acid mixture) and ethanol at three different temperatures (313.15, 323.15, and 333.15 K) by lipase (Novozym R 435) catalyzed. It was investigated under three different reaction systems of ultrasonic (without DES), deep eutectic solvent (DES, choline chloride: glycerol mole ratio = 1:2, without ultrasonic), and ultrasonic-assisted DES. The results showed that the combination of the ultrasonic and DES had a synergistic effect on the enzymatic reaction. The activation energy of the reaction was the smallest (24.82 kJ/mol) compared to the other two processes (ultrasonic: 34.38 kJ/mol and DES: 29.43 kJ/mol), and the reaction was easier to proceed. Subsequently, the reusability of lipase and the composition of the product were studied. The results showed that the conversion rate of fatty acid ethyl ester decreased from the initial 93.33% to 80.31% when the experiment was repeated five times in the ultrasonic-assisted DES system. The ethyl ester composition of the product was mainly concentrated between C 14 and C 24 , and the proportion of ethyl oleate was the most abundant.