The invention relates to a low temperature seedling hardening method for a psammosilene tunicoides tissue culture seedling. The method comprises the steps: transferring psammosilene tunicoides bottled seedling into a seedling hardening culture chamber, and fumigating in a closed manner for 20 min by virtue of potassium permanganate and formaldehyde; culturing the tissue culture seedling for 7 days in an environment with good ventilation at the ambient temperature of 16 to 18 DEG C, the light intensity of 1800 to 2000 Lx and the lighting time of 12 h/d; adjusting the temperature to 11 to 13 DEG C, adjusting the light intensity to 1500 to 1800 Lx, continuously culturing for 14 days, then taking out the psammosilene tunicoides tissue culture seedling by virtue of tweezers, soaking in a 800-time carbendazim disinfectant for 2 h, air-drying, and then transplanting the psammosilene tunicoides tissue culture seedling into a seedling hardening matrix. On the basis that the content of soluble sugar of plants is increased in a low temperature environment, the water content is reduced, the enzyme activity is reduced and air holes are closed, the stress resistance of the plant is finally improved, the rejuvenation period is obviously shortened after the seedling is transplanted, and the probability of the plant infected by root rot and stem rot is reduced, so that the transplanting survival rate is increased to 95 percent or more, and the seedling hardening transplanting survival rate is greatly increased.