Since scaffolds are engineered to support functional tissue formation, their design and materials play an essential role in medical fields by providing different mechanical function. The aim of this study was to investigate the synthesis and structural characterization of collagen-gelatin (COL-GEL) composite scaffolds containing fluorapatite (FA) nanoparticles as well as evaluation of the osteogenic differentiation of human adipose-derived stem cells (hADSCs). First, the composite scaffolds were evaluated using Fourier transform infrared spectroscopy, scanning electron microscopy, and X-ray diffraction. The cytotoxicity of scaffolds and various concentrations of FA nanoparticles was studied through MTT assay and acridine orange/ethidium bromide staining. Next, the differentiated hADSCs were analyzed using Alizarin red and von Kossa staining, calcium content assay, alkaline phosphatase (ALP) activity, real-time RT-PCR, and immunocytochemical analyses. According to the characterization analyses, the composite scaffolds were properly integrated. The results also illustrated that COL-GEL composite scaffolds in the presence of FA nanoparticles not only showed no cytotoxicity but also increased ALP activity and calcium deposition as well as the expression of osteogenic genes, including Runx2, Col-I, ALP, and osteocalcin and the synthesis of proteins such as osteocalcin and osteopontin in vitro. The obtained data were confirmed by Alizarin red and von Kossa staining. These results are very promising for further tissue engineering experiments, in which FA nanoparticle incorporation into COL-GEL composite scaffolds is a novel approach that improves the surface COL-GEL composite scaffolds for tissue engineering application in vitro.