Abstract: ⁹⁹Tc^m-rituximab has been demonstrated to be having favourable properties for sentinel lymph node imaging. The aim of this research was to develop the formulation of the⁹⁹Tc^m-rituximab freeze-dried kit. Firstly the rituximab was modified by 2-Mercapitoethanol, with the glucose as the excipient, and 1 mL phosphate buffers to compose the kit, which was dispensed into nitrogen-filled vials and aliquots frozen at -20 ℃. ⁹⁹Tc^m-rituximab was prepared from the ⁹⁹Tc^m-glucose via ligand exchange method. Both of the labeling yield and the radiochemical purity were more than 90%. The results of biodisdistribution of ⁹⁹Tc^m-rituximab in normal rat with front pad injection showed that the accumulation of the sentinel lymph node increased from 1 h (0.93±0.39%ID) to 4 h (2.14±0.46%ID). The ratio of the uptake of sentinel lymph node to that of the injection site was 14.80±2.11% at 4 h, with no significant change at 18 h (13.03%±3.98%). The SPECT imaging in rat showed that the sentinel lymph node can be seen clearly without the secondary lymph node imaging 30 min~18 h p.i.. In general, the frozen kit of rituximab was feasible to prepare the sentinel imaging agent ⁹⁹Tc^m-rituximab, which possesses satisfied properties for sentinel lymph node SPECT imaging.