Biodiesel and its synthesis process have been increasing in popularity as a potential alternative to fossil fuels. The lipase catalyzed biodiesel synthesis process is considered to be a viable option for industrialization owing to its environmentally friendliness and energy efficiency. However, because of the long reaction duration and low biodiesel yield, it is not yet suitable for large-scale manufacturing. To overcome these limitations, in this paper we presented a novel strategy combined deep eutectic solvents with ultrasonic technique in a scale-up reactor for biodiesel synthesis catalyzed by liquid lipase Yarrowia lipolytica Lipase 2. With the ultrasonic reactor, 93.3% yield biodiesel was obtained under the optimized conditions of 30% water content, 3% lipase loading, 1000 rpm stirring speed and 60 W ultrasonic power in only 6 h reaction time. A kinetic model was built to study the dynamics of the liquid lipase catalyzed reaction system. Then, the model was applied to simulate and optimize the initial water addition and oil-methanol molar ratio in the reactor. The results indicate that the kinetic model fits well for the simulation of this reaction system, which contributes to optimizing the dynamic analysis of the complex system. Meanwhile, a deep eutectic solvent synthesized from natural inexpensive materials was applied for the first time in combination with ultrasonic in scale-up reactor for the liquid lipase-catalytic biodiesel production. With this approach, the biodiesel yield could attain 99% with a reaction time of 6 h and 60 W ultrasonic powers. The reusable batches of lipase were also enhanced, and the biodiesel yield could still reach more than 89% after reusing 5 batches. These results demonstrate that the liquid lipase catalyzed synthesis of biodiesel with the assistance of ultrasound and deep eutectic solvent has a strong potential for industrial application.