BACKGROUND:: Collagen is a common used scaffold in tissue engineering, its specific molecular structure and biological activity are conducive to a variety of cell adhesion, proliferation and differentiation, and can speed up wound healing and degradation to provide sufficient space for new tissues. OBJECTIVE:: To prepare a kind of inhomogeneous double layered collagen composite incorporated with basic fibroblast growth factor (bFGF) loaded chitosan-heparin (CS-Hep) nanoparticles, and to evaluate its biological safety. METHODS:: The air-dried dense layer collagen membranes and freeze-dried loose layer collagen membrane were prepared respectively and then cross-linked by D-ribose (termed as CAM and CFM respectively). CS-Hep nanoparticles were prepared and then dropped on the surface of CFM. The wet CAM was laid on the surface of CFM with nanoparticles, and then air dried completely at 4 °C. The obtained material was termed as bDM. Its physical and chemical properties were investigated. In addition, its bio-safety was also examined through acute systemic toxicity, pyrogen, hemolysis and cellular toxicity tests. RESULTS AND CONCLUSION:: The bFGF/double-layered collagen composite was made up of double layers with one dense layer (CAM) and another loose layer (CFM). As part of bDM, CS-Hep nanopartiles with irregular global shape were aligned in the scope of D-period structure of the inner surface of CFM; the bFGF/double-layered collagen composite had no systemic acute toxicity, no cytotoxicity and pyrogen reactions as well as no hemolytic effect. The bFGF/double-layered collagen composite is prepared and found to have good biocompatibility and safety. Moreover, this material conforms to the ISO 10993-1, and can be used as a basic scaffold material in tissue engineering.Wang JH, Li XM, Tao XJ, Chen HJ, Shi JW, Liu LR, Zhang QQ. Preparation and biological safety of basic fibroblast growth factor/double-layered collagen composite. Zhongguo Zuzhi Gongcheng Yanjiu yu Linchuang Kangfu. 2011;15(47): 8801-8804.[http://www.crter.cn http://en.zglckf.com]