The essential oil from Citrus hystrix leaves is used commercially in Malaysia as a flavor and aroma agent, perfumery, and medical preparations. The main objective for this study was to determine the optimum parameters of ultrasonic pre-treatment; amplitude, particle size of leaves and pre-treatment time prior to the Soxhlet extraction for Citrus hystrix essential oil via Response Surface Methodology (RSM). Fresh Citrus hystrix leaves were dried, blended and sieved to different particle sizes before undergoing ultrasonic pre-treatment and Soxhlet extraction procedure. The ultrasonic pre-treatment was carried out at amplitude ranging from 30% to 70%, particle sizes of 100 μm to 1000 μm, and pre- treatment time of 5 min to 30 min. Soxhlet extraction was conducted for 8 hours with a solid to liquid ratio of 1:20, at the reaction temperature of 68°C using hexane. Results show that the ultrasonic amplitude of 45%, pre-treatment time of 20 minutes, and particle sizes of 200 µm were found to be optimal extraction parameters in achieving maximum oil yield after Soxhlet extraction procedure (6.98 %). Treated leaves yielded 6.54 % of oil extract which was higher than untreated leaves (5.70 %). The functional groups related to the essential oil and powder of Citrus hystrix were limonene, aldehydes, and ester as determined by the Fourier Transform Infra-Red Spectroscopy (FTIR) analysis. The Scanning Electron Microscope (SEM) images reveals that oil glands of treated leaves powder of Citrus hystrix ruptured and cell wall degradation rather than untreated samples. Therefore, ultrasonic pre-treatment could be proposed as a promising pre-treatment method combining with the Soxhlet extraction to improve the essential oil yield of Citrus hystrix.