Abstract: Objective To construct human adipose-derived stem cells (ADSCs) sheet by isolated and purified ADSCs and explore its biological characteristics. Methods Fatty samples were collected from young women's abdomen. Then human ADSCs were obtained by isolating and purifying the samples by collagenase digestion, and then identified using flow cytometry. Human ADSCs were incubated and cultured in complete medium and formed cell-sheet. The morphology and structure of cells sheets were observed by scanning electron microscope (SEM) and the expressions of growth factors and stem-related genes in cell-sheets were detected by qRT-PCR and ELISA. Results Human ADSCs were cultured successfully in vitro, and mesenchymal stem cells markers including CD29, CD44, CD90, CD105 were positively expressed on human ADSCs while CD45 (one of hematopoietic stem cells markers)was negative, which was consistent with ADSCs' characteristics. After osteogenic and adipogenic induction, the calcified nodule and fat drops could be seen using Alizarin red staining and Oil red staining respectively, which suggested that the human ADSCs had multiple differentiation potential. SEM analysis revealed that highly adherent human ADSCs grew in dense layers. The mRNA level of growth factors including TGF-β1, FGF, HGF and VEGF and the expression of stem-related genes like NANOG and OCT4 increased significantly in ADSCs sheets compared with the human ADSCs itself. In addition, the protein level of TGF-β1, FGF2, HGF, VEGF were significantly higher in the cell suspension of cell-sheet by ELISA. Conclusion Human ADSCs cell-sheet is constructed successfully in our study. It can express higher level of some growth factors and stem-related markers, which may provide important evidence for the tissue regeneration and repair using human ADSCs cell-sheet.