Hydroxyapatite (HA) is a highly biocompatible material that has similar properties to human bone. Researchers have adopted different methods for the synthesis of HA from different natural sources. This paper addresses the synthesis method of nano-hydroxyapatite (n-HA) from Hilsha Fishbone. The synthesized n-HA has been characterized by UV, FTIR, TEM, XRD and cytotoxicity analysis. The successful synthesis of n-HA has been confirmed by the peak formation in the UV analysis. The presence of functional groups has been identified by the FTIR analysis. According to the TEM images, the synthesized n-HA is irregular in size and shape. Based on the XRD analysis, the formation of crystals by the n-HA has been assured. The in-vitro cytotoxicity analysis confirmed the bio-safety of the synthesized n-HA. Based on the findings, the synthesized n-HA can be applied in different biomedical applications.