This paper presents the production of biodiesel from Eucalyptus oil using ultrasonic energy and optimization of process parameters by the response surface methodology. The Box-Behnken design has been used to plan the experiments and the response surface methodology (RSM) is selected to develop mathematical relationships between process parameters and desired response (biodiesel yields). The effects of various factors such as ultrasonic frequency, ultrasonic power, molar ratio, reaction temperature, and time on reaction yield were studied to optimize the process. The results showed that all of the considered parameters affect the reaction efficiency significantly. The optimum combination of the findings include: pulse frequency of 29.6 kHz, ultrasonic power of 110.25 W, molar ratio (methanol: oil) of 6.36: 1, reaction temperature of 34.31 degrees C, and reaction time of 7.4 1min that caused the reaction yield of 95.7%. The methyl ester yield was also characterized by physicochemical properties that were found to be in good agreement with ASTM D 6751 and EN 14214 standards.