Microsphere based drug delivery systems show great advantages for tissue engineering. However, it is still a big challenge to fabricate microspheres with capability in delivering and controlled releasing multiple growth factors. In the present study, double-layered microspheres consisting of an inner-layer of small core particles and an outer-layer of big shell particles were developed to sequentially release cell homing factors (SDF-1) and osteoinductive growth factors (BMP-2) for bone regeneration. In vitro release testing showed that bioactivity of both growth factors retained within the microspheres and differential release of SDF-1 and BMP-2 was achieved. Microspheres with both growth factors showed an obvious chemotaxis effect on preosteoblasts by inducing more cell migration. In osteoinductive ability tests, the microspheres with both growth factors showed higher ALP activity and more mineralized modules than control groups after culturing for 2 weeks. The expression of bone development transcription factors (Runx2, OCN, Osterix) as well as Smad signals (Smad 1, 5, 8) showed higher gene expression in the dual growth factor group. Our results suggest that a double-layered microsphere system enhances the recruitment of osteogenic cells and osteoinduction, which provides a promising platform for bone regeneration.