The paper aims to develop a rapid propagation system for the medicinal resource Rubus chingii var. suavissimus. Using tender stem segments with buds as explants, the study investigated the effects of sterilization duration, hormone concentration ratios in subculture proliferation media and rooting media, as well as transplanting substrate ratios on aseptic line establishment, cluster bud proliferation, test-tube plantlet rooting, and transplant growth of tissue-cultured seedlings. Orthogonal test design combined with stepwise regression analysis was used to screen the optimal conditions of each link. Results showed that the optimal sterilization method for explants was treatment with 0.1% HgCl₂ solution for 7 minutes, achieving a survival rate of 80%. The optimal subculture proliferation medium was MS+ 6-BA 1.0 mg/L+ NAA 0.1 mg/L, with a proliferation coefficient of 27.33. The optimal rooting medium was 1/2MS+ IBA 0.5 mg/L, resulting in 100% rooting rate, approximately 13 average roots per plant, and average root length of 1.74 cm. The optimal transplanting substrate ratio was vermiculite: nutrient soil: coconut coir=1:1:1, with a survival rate as high as 86.3%. The successful establishment of a rapid tissue culture propagation system for Rubus chingii var. suavissimus holds significant importance for the sustainable development of sweet tea resources and the conservation of genetic diversity in characteristic medicinal plants.