Cryoablation techniques are effective for the treatment of solid tumors, but generally suffer from the limited cooling effect and low target killing efficiency. To improve the efficacy, the use of ultrasound-induced nucleation was proposed as a new adjunct. The effects of different induced nucleation temperatures on the cell viability at two cooling rates were investigated, and the morphology of extracellular ice crystals, intracellular ice crystal generation and cell damage after rewarming were observed and recorded. The results show that the use of ultrasound-induced nucleation as an adjunct to cryoablation can improve the lethality of tumor cells. The slow freezing group with ultrasound-induced nucleation have the best results with more complete ablation and lower cell viability of 15.60%±2.60%. The rapid freezing group need to be coupled with a lower induced nucleation temperature to better kill the cells. The design and development of cryoablation devices with ultrasound-induced nucleation have practical implications.