Detaching cells from the surface of the flask is a necessary operation for the adherent cell passage. Trypsinization is currently the primary method for cell detachment. However, this method is complicated to automated cell culture. Trypsin can cause irreversible damage to cellular proteins, decreasing of cell yield and long self-repair time. Ultrasonic detachment technology uses physical means to complete cell detachment operations, which can avoid damage to cellular proteins and make the realization of automated detachment easier. In this paper, we have developed a device for ultrasonic detachment. Human umbilical cord mesenchymal stem cells were used to study ultrasonic detachment technology control parameters. The experiment results showed that, the optimum cell detachment was achieved (95% detachment rate and 70% survival rate) in 12 mL 5%-saline with 255 V and 159～161 kHz sweeping input signal, 2 min stimulation period, and 3 mm space between flask s urface and transducer.