In this study, nanocomposite microspheres based on chitosan/gelatin/nanohydroxyapatite were fabricated, and effects of the nanohydroxyapatite/biopolymer (chitosan/gelatin) weight ratio (nHA/P), stirring rate, chitosan concentration and biopolymer concentration on the particle size, and morphology of nanocomposite microspheres were investigated. Particle size of microspheres was modeled by design of experiments using the surface response method. Particle size, morphology of microspheres, and distribution of nanoparticles within the composite microspheres were evaluated using an optical microscope, scanning electron microscopy (SEM) and transmission electron microscopy (TEM), respectively. X-ray diffraction and Fourier transform infrared spectroscopy were applied to study the physical and chemical characteristics of microspheres. Results showed that by modulating the nHA/P ratio, chitosan concentration, polymer concentration, and stirring rate, it is possible to fabricate microspheres in wide rages of particle size (5–150 μm). Analysis of variance confirmed that the modified quadratic model can be used to predict the particle size of nanocomposite microspheres within the design space. SEM studies showed that microspheres with different compositions had totally different morphologies from dense morphologies to porous ones. TEM images demonstrated that nanoparticles were distributed uniformly within the polymeric matrix. MTT assay and cell culture studies showed that microspheres with different compositions possessed good biocompatibility. © 2012 Wiley Periodicals, Inc. J Biomed Mater Res Part A, 2013.