Hosta plants have high value in landscape and pharmacology. Hosta plants in the wild are vulnerable to natural disasters, viruses, diseases and insect pests, and the normal tissue culture is faced with high maintenance costs, and chances of pollution. Considering the need for breeding new varieties and maintaining the genetic stability, it is of great significance to establish a preservation technique with safety, low maintenance and low consumption. This research was conducted to obtain the optimal in vitro preservation protocol for Hosta plantaginea by using the orthogonal design and to study the effects of different concentrations of sucrose, mannitol, abscisic acid and CCC (chlormequat chloride) in the culture medium on the survival percentages and stability during the slow growth of in vitro plantlets of Hosta plantaginea. The results showed that 100% survival of in vitro Hosta plantaginea plantlets was obtained after six-month storage at 20-25℃, which were cultured in the MS medium supplemented with 50 g/L sucrose, 20 g/L mannitol, 1mg/L ABA, 20 mg/L CCC and 0.7% agar (pH 5.8), and the storage life without subculture was twice as much as that of the control of 3 months on normal medium. The activities of SOD, POD and CAT in preserved in vitro plantlets were higher than those of the control group, and the content of MDA was lower than that of the control group, which indicated that the activity of antioxidant enzymes increased, the degree of lipid oxidation decreased, the senescence process was delayed, and the storage life was prolonged.