The control parameters of the removal of Chlorella pyrenoidosa, which was irradiated by low frequency ultrasonic, is optimized by using single factor experiments and response surface methodology (RSM). First of all, the approximate ranges of the ultrasonic frequency, the ultrasonic power and the irradiation time were estimated with single factor experiments for the further experiments. And then the optimized values of the three control parameters were determined, which were analyzed by using central composite design (CCD) and RSM. The results showed that the removal rate of chlorophyll-a could reach to 64.1% after the irradiation for 6.34min by using ultrasonic of 77.7 kHz and 250W. Ultrasonic technology can remove Chlorella pyrenoidosa cells in water quickly and effectively, so as to achieve the purpose of water purification.