The invention relates to the technical field of biology, in particular to an in-vitro regeneration culture method of rhizoma polygonati leaves. The in-vitro regeneration culture method comprises the steps of explant selection, adventitious bud induction, cluster bud multiplication, rooting culture as well as seedling hardening and transplanting. In the technical scheme of the invention, strip-shaped big seedlings or fine needle-shaped small buds in a multiplication stage during tissue culture are selected as explants; by preparing a brand-new culture medium, breeding of rhizoma polygonati canbe quickly realized, repeatability is high, materials are easy to obtain, the operation is simple and convenient, the breeding efficiency is high, the bottleneck problem of singly using rootstocks andapical buds to breed a regenerative material during the tissue culture of the rhizomal polygonati is greatly broken through, the problems of germplasm degeneration of the rhizomal polygonati as wellas long period, low breeding rate and the like in conventional breeding are fundamentally solved, and a foundation is laid for meeting the market demands, relieving ecological stress and industrializing and normalizing nursery stock production.