Background: Astragalus membranaceus Mongholicus (Astragalus) is one of the most widely used medicinal plants in the world. It is a traditional Chinese medicine plant that has been used to treat various human diseases and conditions, such as inflammation, gastrointestinal bacterial infections and malignant tumors. With the expansion of artificial planting areas, the demand for high-quality Astragalus seedlings is also increasing. The traditional seedling-raising methods of direct seeding and induced callus have problems such as a long growth period, unstable genetic traits, strict sampling and rooting difficulties. Therefore, it is very important to multiply the excellent germplasm by optimizing the tissue culture regeneration system. Methods: In this study, Astragalus field cultivated plants, which are easily available, were used as materials and the method of tissue culture cuttings was used to select the direct inoculation method of stem segments as explant materials to establish a plant regeneration system of Astragalus and the best sampling period, sampling site and optimal medium for regeneration of field cultivated plants were screened. Result: The best sampling period for plant regeneration of Astragalus in the field was about 40 d after the emergence of the 3-year-old Astragalus and the rooting rate of the regenerated plants was 100.00%. The best sampling site is the middle section of the stem and the regeneration system using the middle section of the stem as the explant is better than using the top and bottom of the stem as the explant and the rooting rate of the regenerated plants is 100.00%; the best medium for the regenerated plants is N22: 1/2MS+1 mg/L NAA and T28: 1/2MS+1 mg/L ABT, the rooting rates were 76.67% and 96.67%, respectively. The regenerated plants established with the stems of the field cultivated plants of Astragalus as explants were all able to induce seedlings after 1 week and the regenerated seedlings start to take root after 3 weeks of growth and can be transplanted and propagated after 6 weeks of culture. The regeneration system established in this study by direct cuttings from cultivated Astragalus stems is more rapid and efficient, breaking through the difficulties of limited access to material and long growth period of regenerated seedlings in the traditional regeneration system and providing technical support for the conservation and utilization of wild resources of Astragalus and rapid expansion of new varieties and germplasm conservation.