The invention discloses an isolation extraction and culture method for human adipose-derived stem cells (ADSCs) for a clinical back-transfusion grade cell therapy, belongs to the field of biological stem cells medical science and medicine, and particularly discloses a method for in-vitro isolation, large-scale culture and amplification and storage of human adipose-derived mesenchymal stem cells (ADSCs).According to the method, target adipose mesenchymal stem cells are locked through high-fitting in-vitro extracellular matrix adhesion, attached culture is conducted, and by means of an optimized culture medium combination, an in-vivo imitation ecological environment serum-free domesticated culture system is graded, and the adipose-derived mesenchymal stem cells with the clinical therapy grade are obtained.The obtained stem cells meet the standards of International Society For Cellular Therapy (ISCT) and have high purity and strong stem cell proliferation and amplification and passaging activity, and the technology of protecting cell activity through frozen preservation is achieved; the method is a core foundation in the field of stem cell therapies, and the requirements of clinical drug design and development of the stem cells and safe and effective application of stem cell therapy back transfusion carried by medical institutions are met.