This study investigated the fabrication and modification of gelatin microspheres via single emulsion method and silane coupling agents, respectively. Herein, the influence of oil phase and washing procedure on morphology and uniformity of microspheres was evaluated. The results of field emission scanning electron microscopy indicated a positive effect of olive oil on the production of uniform and approximately mono‐size spheres while monodispersity increased after samples were washed under ultrasonication. Investigation of crosslinking degree demonstrated that GPTMS could not show efficiency as high as glutaraldehyde, but the lack of toxicity and inducing bioactive behavior to the microspheres has made them appropriate for bone applications. Moreover, the stability of spheres after 14 days in phosphate buffered saline proved the ability of (3‐glycidyloxypropyl)trimethoxysilane to crosslink gelatin. The presence of silane coupling agents in prepared spheres caused the biomimetic formation of hydroxyapatite after a 14‐day immersion in simulated body fluid as observed by field emission scanning electron microscopy‐energy‐dispersive X‐ray spectroscopy, changes in pH, Fourier transform infrared spectroscopy, X‐ray diffraction, and atomic force microscopy. Adhesion, spreading, and proliferation of L929 cells on the hybrid microspheres corroborated the potential of gelatin spheres for biomedical application. © 2018 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2018, 135, 46739.