Preservation of living lung cancer tissues will provide more complete sample information for in vitro experimental studies, such as lung cancer genetic screening and targeted drug screening. This article studied the vitrification method of living lung cancer tissue. Firstly, the needle immersion method was used to vitrify a single piece of lung cancer tissue, and the concentration and equilibrium time of the required cryoprotectant were optimized. Secondly, multiple lung cancer tissues were vitrified in cryotubes, and the volume of cryoprotectant solution and equilibrium time were optimized. Finally, the effects of the traditional slow freezing, rapid freezing without cryoprotectant, and vitrification methods were compared, and low-temperature microscope was used to analyze the damage mechanism of ice crystal. The results showed when 20% EG+20% DMSO+0.5 mol/L trehalose was used as the cryoprotectant, the equilibration solution and the vitrification solution are loaded for 3 min and 1 min, respectively, the needle immersion method and vitrification in a 0.25 ml cryotube have the highest tissue viability (79.96% and 80.44%) after recovery. Immunohistochemistry showed that the tissue structure of lung cancer after vitrification was less damaged, only a few positive expression of TUNEL in the cells, compared with slow freezing and fast freezing without cryoprotectants. Low-temperature microscopy showed that only a few small ice crystals appeared in and around the vitrified tissue, while obvious ice crystals appeared in slow freezing and rapid freezing.