It is advantageous to incorporate controlled growth factor delivery into tissue engineering strategies. The purpose of the present study was to develop a novel tissue engineering scaffold with the capability of controlled releasing BMP-2-derived synthetic peptide. Porous nano-hydroxyapatite/collagen/poly(L-lactic acid)/chitosan microspheres (nHAC/PLLA/CMs) composite scaffolds containing different quantities of chitosan microspheres (CMs) were prepared by a thermally induced phase separation method. Dioxane was used as the solvent for PLLA. Introduction of less than 30% of CMs (on PLLA weight basis) did not remarkably affect the morphology and porosity of the nHAC/PLLA/CMs scaffolds. However, as the microspheres contents increased to 50%, the porosity of the composite decreased rapidly. The compressive modulus of the composite scaffolds increased from 15.4 to 25.5 MPa, while the compressive strength increased from 1.42 to 1.63 MPa as the microspheres contents increased from 0% to 50%. The hydrolytic degradation and synthetic peptide release kinetics in vitro were investigated by incubation in phosphate buffered saline solution (pH 7.4). The results indicated that the degradation rate of the scaffolds was increased with the enhancement of CMs dosage. The synthetic peptide was released in a temporally controlled manner, depending on the degradation of both incorporated chitosan microspheres and PLLA matrix. In vitro bioactivity assay revealed that the encapsulated synthetic peptide was biologically active as evidenced by stimulation of rabbit marrow mesenchymal stem cells (MSCs) alkaline phosphatase (ALP) activity. The successful microspheres-scaffold system offers a new delivery method of growth factors and a novel scaffold design for bone regeneration.