Objective To observe the growth of human adipose-derived stem cells （hASCs） in bladder acellular matrix graft-silk fibroin （BAMG-SF） bilayer scaffold and to analyze the biological compatibility of BAMG-SF with hASCs. Methods hASCs were isolated from human subcutaneous adipose tissue after collagenase digesting, filtrating and centrifuging, then cultured in the leaching solution of BAMG-SF. The cytotoxicity of scaffold was evaluated by CCK-8 cell viability assay, and the growth curves were also observed. Surface morphology on BAMG-SF was observed by scanning electron microscopy （SEM）. The hASCs of passage 3 were seeded onto the BAMG-SF bilayer scaffolds for 1 week, then the BAMG-SF bilayer scaffolds seeded with hASCs were transplanted into nude mouse for 1 week or 2 weeks. The growth of cells in BAMG-SF biomaterials was observed by HE staining. The species origin of these cells in the BAMG-SF scaffolds cultured in vivo was detected by Immunofluorescence. Results hASCs maintained high proliferation rate in the leaching solution of BAMG-SF and the BAMG-SF scaffolds were nontoxic absolutely. According to the growth curves of hASCs cultured in the leaching solution of the BAMG-SF and DMEM, BAMG-SF scaffolds were conducive to the growth of hASCs. The histological study found that hASCs could grow into the space of the BAMG-SF scaffolds after cultured in vitro and in vivo. There were more cells in the scaffolds cultured in vivo than in vitro. Immuno-fluorescence suggested that some of the cells inside the scaffolds were hASCs. Conclusion BAMG-SF bilayer scaffolds are nontoxic and have a good biocompatibility with hASCs, which can be used as a vehicle for hASCs in bladder defect reconstruction.