Abstract: The preparation of biodiesel from inedible low-cost acid oil with a high average acid value(AV) of about 33.07 mgKOH/g via pre-esterification and transesterification processes in a self-designed continuous biodiesel production apparatus assisted by multi-frequency ultrasonic at high efficiency and low energy consumption was investigated. The influences of operating variables including flow rate of material(residence time), ultrasonic power and frequency or combinations, the amount of catalyst, the molar ratio of methanol to oil at ambient temperature and the specific energy consumption on the transesterification reaction were discussed. The results show that multi-frequency ultrasonic irradiation is superior to single-frequency in enhancing biodiesel production process; the yield of fatty acid methyl esters(FAME) about 96.83% is attained with 15-25-35-40 kHz of frequency combination, 200 W of powers, 25 L/h of flow rates of the processed oil (residence time 54 min), 6:1 mol ratio of methanol to oil,and 1.2% KOH (% of acid oil). 50 L waste acid oil can be converted into 48 L biodiesel that meet the standards GB19147-2009, and the overall electric power and time consumptions are about 5.42 kWh and 8.667 h, respectively.