Ultrasonic treatment was applied to enhance the biomass and lipid accumulation of mixotrophic microalgae. The optimal microalgal ultrasonic power, ultrasonic frequency, ultrasonic interval and growth phase were 20 W, 20 Hz, 2 s and logarithmic phase, respectively. The maximum biomass concentration and lipid content reached 2.78 g L-1 and 28.5%, which were 26.9% and 37% higher than those of the control group. Microscope analysis shows that ultrasonic exposure caused tiny cracks or holes on the surface of cell walls, but did not damage the integrity of algal cell structure. After ultrasonic stimulation, the permeability of membrane and the transport of nutrients were improved, and the utilization rate of substrate and pigment concentration increased 22.7% and 18.4%, respectively. However, excessive ultrasonic irradiation significantly inhibited the cell growth and lipid accumulation of microalgae. This study indicates the feasibility and efficiency of using low-strength ultrasound in promoting biomass and lipid production of microalgae.