In this study, the vitrificated plants of hybrid aspen (Populus tremula× P.tremuloides) by micropropagation were used as the materials,and ELISA method was adopted to measure the content of endogenous plant hormones in the plants. At the same time, the factors that influenced the vitrification of the plant as well as de-vitrification method were also studied by changing medium’s component, which was to provide useful information for commercial propagation of good hybrid clones of aspen. The results were as follows: 1)The ABA and IAA contents in the vitrificated plants were higher than those in the non-vitrificated plants obviously, and the GA content was little lower than that of the non-vitrificated plants. Therefore, the higher IAA or ABA content in the vitrificated plants was possibly the main reason for the vitrification of the plant.2)The ratios of endogenous plant hormones such as ABA/GA and IAA/GA in the vitrificated plants of hybrid aspen by tissue culture were higher than those in the non-vitrificated plants, which indicated that the equilibrium relationship among different endogenous hormones in vitrificated plants was deviated from the normal, then possibly resulting in the vitrification of the plant.