A549 cell is a good model for screening apoptosis-inducing drugs for lung adenocarcinoma in vitro research. It is of great significance to maintain high-activity and high-quality A549 cells in the biobank. In the paper, a non-contact ice-seeding slow cooling program were adopted to reduce the subcooling of the cryoprotectant(CPA) during the cooling process. The concentration of EG, Me₂SO and trehalose were optimized for the cryopreservation of A549 cells. The results show that the non-contact ice-seeding cooling program can effectively reduce the subcooling of the cryoprotectant compared to the traditional slow cooling program; the optimized cryoprotectant combination is 7.5%(V/V)Me₂SO+2.5%(V/V)EG+200 mmol trehalose. After cryopreservation and rewarming, the recovery rate of the cells using this CPA is higher than the traditional cryoprotectant of 10%(V/V) Me₂SO (91.59±1.51% vs 86.72±1.86%). The proportion of normal cells is high, which indicates that the combined cryoprotectant can effectively inhibit apoptosis after cryopreservation.