Nano-hydroxyapatite (nano-HAp) is significant import as a bio-ceramic material for use in orthopedic applications because of its comparability in size, crystallography and substance creation with human hard tissue. In this present study, the biologically high interest of nano-HAp was synthesized as green template mode approaches using the various fatty acids like linoleic acid, lauric acid, and oleic acid. The influence of the fatty acid for the formations nanoHAp structure and nature of fatty acid during synthesis was investigated. The nano-HAp functionality, crystallinity, and morphology were determined by FTIR, XRD, and SEM techniques, respectively. The biocompatibility of HAp on MG63 cell line was confirmed by in vitro MTT assay method. The in-vitro studies showed better biocompatibility and cell proliferation results on the template assist synthesized nHAP. These results indicate that nHAp form the green methodology could be a promising material for bone tissue engineering.