Abstract: Objective To explore the method of preparing ⁹⁹Tc^m-MIBI liposome nanoparticles, investigate its' physical, biological characterization and stability in vitro, and study its biological distribution characteristics in mice. Methods The ⁹⁹Tc^m-MIBI liposome nanoparticles were prepared by the ethanol infusion-ultrasonic dispersion process. The particle size and encapsulation efficiency were measured. The radioactivity purity and stability of ⁹⁹Tc^m-MIBI liposome nanoparticles in normal human serum and normal saline(NS) at different time points were observed under the condition of 37℃ in vitro. The distribution characteristics of 15, 60, and 120 min in mice were studied. Results The ⁹⁹Tc^m-MIBI liposome nanoparticles prepared by ethanol infusion-ultrasonic dispersion method were spherical and homogeneous under the electron microscope, with an average diameter of (168.2±18.6) nm. In vitro stability test showed that ⁹⁹Tc^m-MIBI liposome nanoparticles incubated 15, 30, 60, 120 min in normal serum and NS respectively, and their radiochemical purity was 96%, 93%, 90%, 89% and 92%, 89%, 86% and 85%, respectively. In vivo biodistribution experiment showed that compared with 99mTc-MIBI, after intravenous injection of ⁹⁹Tc^m-MIBI liposome nanoparticles, the spleen uptake was significantly observed and the radioactivity uptake rate of kidney was low during the observation time. Conclusion The preparation method of ⁹⁹Tc^m-MIBI liposome nanoparticles is simple and has ideal physical, biological characterization, and the stability in serum is good. Compared with ⁹⁹Tc^m-MIBI the kidney uptake rate was low, and the circulation time in vivo of the mice was prolonged.