The effects of several critical factors including the amount of EG, the reaction time, the concentrations of Ca(CH3COO)2 and NaHCO3, the concentration of H3PO4, and the amount of acetic acid were investigated for synthesizing hollow mesoporous hydroxyapatite nanoparticles (hm-HANPs). We optimized the reaction conditions as follows: the concentration of Ca(CH3COO)2 and NaHCO3 was 0.3 M, the ratio of Ca(CH3COO)2 and NaHCO3 to ethylene glycol (EG) was 1:5, the reaction time for the formation of hydroxyapatite was 3 hr, the concentration of H3PO4 was 0.01 M, and the ratio of hm-HANP solution to acetic acid was 1:3.125. Monodispersed hm-HANPs with a defined particle size could be obtained. A fluorescent dye named fluorescein isothiocyanate (FITC) was successfully linked to the surface of hm-HANPs for the bioimaging of hm-HANPs in living breast cancer cells (BT-20). The FITC-hm-HANPs were taken up by the cells and stayed in the cytoplasm with high biocompatibility. The information obtained in this study will benefit the synthesis of hydroxyapatite nanostructures for many biomedical-related applications.