In the present investigation, we have successfully synthesized luminescent Eu -doped and Eu /Sr codoped hydroxyapatite (HA) nanoparticles through sol-gel assisted precipitation method with the aim of developing novel biomaterials containing osteoblast mineral (Sr ) and luminescence activator (Eu ). The structure, morphology, thermal stability, and luminescence properties of the resultant spherical nanoparticles (50-100 nm diameters) were studied. Moreover, the in-vitro bioactivity of Eu Sr HA nanoparticles was investigated by immersing in the simulated body fluid for many weeks. The antimicrobial activity results against gram positive and gram negative bacterial stains, showed better resistivity for the Eu Sr HA among the other compositions. The MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay of live/dead cells cultured with Eu /Sr -doped HA nanoparticles retained its normal morphology and did not show a significant impact on cell proliferation at various incubation days, which evidence for the material's superior biocompatible nature even at a higher concentration of 375 µg/mL. Thus, the incorporation of dual ions in HA nanoparticles with strong luminescence properties develops potential biomaterial for live cell imaging and in nanomedicine. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 106B: 2191-2201, 2018.