An aqueous enzymatic-ultrasonication extraction (AEUE) technique involving sequential treatment with enzyme mixtures (neutral protease/cellulase/pectinase) followed by ultrasonication was proposed for the extraction of oil from Sapindus mukorossi seed kernels. Statistical optimization was successfully applied to determine the optimal AEUE conditions. Three parameters, including enzyme amount, ultrasonic time and power, were screened from eight parameters using the Plackett-Burman design and further optimized by the Box-Behnken design for the attainment of a high yield of S. mukorossi seed kernels oil. The optimal AEUE conditions with a yield of 82.67% +/- 2.21%, relative to the Soxhlet method, were obtained as follows: incubation time of 8 h, amount of 4%, pH of 7, incubation temperature of 60 degrees C, shaking speed of 600 rpm, water solid ratio of 16 mL/g, ultrasonic time of 56 min, and ultrasonic power of 240 W. The S. mukorossi seed kernels oil acquired using AEUE exhibited a lower acid value and a higher iodine value than that acquired by the Soxhlet method. Meanwhile, all the oils showed similar fatty acid profiles according to GC-MS analysis, regardless of the extraction method, indicating that AEUE is capable of S. mukorossi seed kernels oil extraction. In summary, the results from this study indicate that the developed AEUE is an efficient process for the separation of nonedible oil of for use in biodiesel production.