In order to explore the variation law of rejuvenation based on tissue culture, we selected 10yearold Pyrus betulaefolia for continuous subculture, and then counted the propagation coefficient and rooting rate of multiple shoots of different subculture passages. We also observed and recorded the morphological changes of leaves and measured the content of endogenous hormones. The results showed that: (1) the rooting rate of multiple shoots increased from 0 to 66.70% and the propagation coefficient increased from 2.13 in the first subculture to 4.20 through continuous subculture. (2) The leaves showed a lobes at 3rd subculture and then the degree of lobes gradually deepened; during the continuous subculture, leaf area and vein number of multiple shoots were decreased significantly, and leaf perimeter and leaf index showed a trend of decreasing firstly and then increasing. (3) The endogenous IAA content of multiple shoots reached 46.39 ng·g^-1 at 6th subculture, which was significantly higher than that of 0th subculture; with the increase of subculture passages, the endogenous ZR showed a trend of increasing firstly and then decreasing, and the endogenous GA₃ content did not change significantly, while the endogenous ABA content gradually decreased in the leaves; the values of IAA/ABA and IAA/ZR in the leaves increased with the number of subculture. (4) The ABA content and IAA/ZR of multiple shoots leaves showed significantly negative correlation and significantly positive correlation with rooting rate. The number of leaf lobes and IAA/ABA showed a significantly positive correlation with rooting rate, while the number of leaf veins showed a significantly negative correlation with rooting rate. It can be seen that continuous subculture can significantly improve the rooting ability of P. betulaefolia multiple shoots, and it is closely related to leaf morphology and hormone content and ratio of multiple shoots, which provides reference for vegetative propagation of difficult rooting plants.