Sentinel lymph node detection is widely used to identify lymph nodes that receive lymphatic drainage from a primary tumor. Tc-99m labeled iron oxide nanoparticles were prepared to invent a new colorful radioactive agent for sentinel lymph node detection. Iron oxide nanoparticles were produced by co-precipitation of FeCl3 and FeCl2 in the presence of NaOH. Then iron oxide nanoparticles were labeled with Tc-99m. Tc-99m labeled nanoparticles (7.4 MBq/0.1 mL) were intradermally injected in the distal hind limb of 16 rabbits. Dynamic and static lymphoscintigraphic images were taken for 24 h. Labeling efficiencies of Tc-99m-iron oxide nanoparticles were over 99%. Their sizes are between 50 and 60 nm. Tc-99m-iron oxide nanoparticles were accumulated in the popliteal lymph node in 11 of 16 rabbits (69%). Retention of nanoparticles in the popliteal lymph node was obvious at from 2nd through 24th hours. The radioactive lymph node was identified easily by gamma probe. The popliteal lymph node was excised and established for radioactivity and black dye. These black and radioactive nanoparticles may be potential agent successfully used for sentinel lymph node detection.