The introduction of biomineralization was coupled with the co-precipitation synthesis process of nano-hydroxyapatite with the addition of chondroitin sulfate as a template agent. The effect of a variety of processing conditions on the properties of final hydroxyapatite (HA) product was investigated by orthogonal design. The ratio of calcium to phosphorus was detected by chemical analysis, the phase composition was evaluated by X-ray diffraction (XRD), and the powder morphology was characterized by transmission electron microscope (TEM). The process scheme, moreover, was optimized by the analysis of four aspects which may have different extent of influence on product properties. It can be concluded from the results that product properties can be affected remarkably by the content of chondroitin sulfate and the pH value of reactant, less remarkably by the reaction temperature and slightly by the reaction time.