Titanium (Ti) and its alloys are the most attractive metallic biomaterials since their first introduction to the biomedical area. Their major drawback is that they do not promote complete osteointegration due to their low bioactivity; thus, it is necessary to perform a surface treatment or apply a surface coating to increase the initial formation of surrounding bone tissue. In this study, strontium (Sr)-doped hydroxyapatite (HA) coatings on Ti6Al4V substrates were prepared by biomimetic method in which 2 times concentrated simulated body fluid (2xSBF) solution was used as the apatite growth medium. The characterization studies with X-ray diffraction (XRD) and scanning electron microscopy (SEM) indicated that as the amount of strontium ion (Sr2+) replacing with calcium ion (Ca2+) in the structure increases, crystallinity decreases without causing a significant change in morphology of the HA deposits. Incorporation of Sr2+ ions resulted in the disappearance of Fourier transform infrared (FTIR) bands of carbonate ion (CO32-) which was found in the structure of pure HA biomimetic coating. The biological results demonstrated that Sr-doped HAs presented no cytotoxicity on Saos-2 cells. This study showed that the incorporation of Sr into apatitic structure is possible by mimicking the natural route and this procedure yields a promising bioactive coating on Ti alloys to be used in bone replacement therapies especially associated with osteoporosis.