To overcome clinical issues caused by large bone defects and subsequent nonunion, various approaches to bone regeneration have been researched, including tissue engineering, biomaterials, stem cells and drug screening. Previously, we developed a free-standing biodegradable polymer nanosheet composed of poly(L-lactic acid) (PLLA) using a simple fabrication process consisting of spin-coating and peeling techniques. We reported that sandwich-type PLLA nanosheets loaded with recombinant human bone morphogenetic protein-2 (rhBMP-2) displayed long-lasting, sustained release of rhBMP-2, and markedly enhanced bone regeneration in mouse calvarial bone defects. Here, we fabricated multi-layered nanosheets loaded with fibroblast growth factor-2 (FGF-2), and investigated their application for long bone regeneration. Subcutaneously implanted tri-layered PLLA nanosheets displayed sustained release of loaded rhFGF-2 for about 2 weeks. Next, we prepared critical-sized mouse femoral defects and implanted mono- or tri-layered nanosheets, or a gelatin hydrogel with rhFGF-2. Amongst these conditions, the tri-layered nanosheet most efficiently induced bone regeneration. Indeed, bone regeneration was enhanced even after 4 weeks in the tri-layered nanosheet group, and was accompanied by FGFR1 activation and subsequent osteoblast differentiation. Multi-layered PLLA nanosheets loaded with rhFGF-2 may be useful for bone regenerative medicine. Furthermore, the multi-layered PLLA nanosheet structure may potentially be applied as a potent sustained-release carrier. STATEMENTS OF SIGNIFICANCE: Here, we describe multi-layered poly(L-lactic acid) (PLLA) nanosheets loaded with recombinant human fibroblast growth factor-2 (rhFGF-2) as a modified sustained-release carrier for bone regeneration. In vivo imaging system analysis revealed that subcutaneously implanted tri-layered PLLA nanosheets displayed sustained release of loaded rhFGF-2 for 2 weeks. In critical-sized mouse femoral defects, tri-layered nanosheets loaded with rhFGF-2 most efficiently induced bone regeneration. Notably, bone regeneration was enhanced even after 4 weeks in the tri-layered nanosheet group, and was accompanied by FGFR1 activation and subsequent osteoblast differentiation. Multi-layered PLLA nanosheets loaded with rhFGF-2 may be useful for bone regenerative medicine. Furthermore, the multi-layered PLLA nanosheet structure may potentially be applied as a potent sustained-release carrier.