The hydroxyapatite nanoparticles (nHAPs) were synthesized rapidly by the self-assembled dual-frequency ultrasonic method. The ultrasonic time and power effect on the morphology and phase composition of nHAPs were investigated through field-emission scanning electron microscopy (FE-SEM), X-ray diffraction, energy dispersive spectrometer (EDS) spectrometer, and Fourier transform infrared spectroscopy, which showed that the most uniform nanoparticles were obtained when the ultrasonic time was 30 min and the ultrasonic power was 280 W. Cytotoxicity and hemolysis tests showed that an indistinctive cytotoxic effect was within the concentration of 25–400 μg/mL and the hemolytic ratio was below 2.0% at concentration of 25–200 μg/mL, respectively, revealing a good biocompatibility of nHAPs. By loading tetracycline hydrochloride onto nHAPs spheres, the drug release results showed that the drug loading and encapsulation efficiency were (26.34 ± 2.99)% and (52.68 ± 5.98)%, respectively. The drug-loaded sample shows a slow-release property, indicating that nHAPs may be promising as drug carriers.