Exogenous administration of growth factors has been identified as a potential therapeutic approach for healing wounds. A way to enhance the efficacy of growth factors would be to achieve spatiotemporal control over their delivery to desired sites for an extended period. In this study, we designed and prepared a kind of double-layered collagen membrane, a dense layer and a loose layer, which incorporated basic fibroblast growth factor (bFGF)-loaded chitosan-heparin nanoparticles. The nanoparticles were prepared by polyelectrolyte gelation process and then were sandwiched between the two layers of collagen membrane. The release of model protein human serum albumin (HSA) from the double-layered membrane was tracked by radio-label assay, and the bioactivity of the growth factor on fibroblast cell (L929) was evaluated by MTT assay. The release of protein displayed a spatiotemporal control model and its release in undesired direction was lessened. The bFGF maintained the bioactivities after release from the membranes. Moreover, different release amounts of bFGF from the different layers of the membrane induced significant difference in cell proliferation when the cells were seeded on the different layers of membrane in vitro. This kind of double-layered collagen membrane could have potential applications in the field of tissue repair due to the spatiotemporal control over growth factor delivery, the mild fabrication conditions, and the simple processes. © 2011 Wiley Periodicals, Inc. J Biomed Mater Res Part A, 2012.