Spheroid hydroxyapatite nanoparticles were successfully prepared by titrating Ca(NO3)2•4H2O and (NH4)2HPO4 aqueous solution in the reverse microemulsion, which consists of mixed OP-10(surfactant), cyclohexane(oil phase), and isobutanol (cosurfactant).The structure and morphology of the prepared powders were characterized by means of X-ray diffraction (XRD) and transmission electron microscope (TEM). The optimum composition was investigated via the analysis of the aqueous solution conductivity, and the mechanism of aqueous reaction. The effect of the pH value and the amount of surfactant on the particle size were studied. The results indicate that the best conditions are which the concentration of OP-10 and isobutanol both are 0.2 M, when the water content is between 130 ml-250 ml,it results in pure nano-hydroxyapatite spheroid-like powders, and the water content is about 200 ml, whose diameters are 50 nm-80 nm, and good particle diameter distribution, the particle microstructure is homogeneous, good crystal structure and higher crystallinity.