Abstract: To investigate the role of regulation, uptake and distribution of miR-155 targeted radiolabeled probes in MCF-7 cell line and to provide an effective candidate probe for future in vivo studies. MiR-155 targeted or nonsense control probe was radiolabeled with technetium-99m (^99mTc) using bifunctional chelator NHS-MAG3. The serum stability was evaluated by the Mini-Scan thin layer chromatography. After transfected in MCF-7 cells, miR-155 targeted probe was evaluated by western blotting. Its cellular uptake was measured at different time points. Further, the distribution of fluorescent protein labeled FAM and nonsense probes were evaluated in MCF-7 cells by confocal microscopy. The radiolabeled efficiency of ^99mTc-labeled miR-155 targeted probe was 97%, radiochemical purity was greater than 98%, and radioactive specific activity was 3.75 GBq/μg. In fresh human serum for 12 h, its radiochemical purity was greater than 95%. Compared with untreated cells, unlabeled and labeled miR-155 targeted probes up-regulated the expression of C/EBPβ protein in MCF-7 cells. The cellular uptake of ^99mTc-labeled miR-155 targeted probe was significantly higher than that of untreated cells in MCF-7 tumor cells. Furthermore, the fluorescence-labeled miR-155 targeted probe showed a stable and efficient targeting distribution in MCF-7 cells at 24 h. Therefore, miR-155 targeted radiolabeled probe has good targeting and biological activity at the cellular level in MCF-7 cells, and has potential application value for in vivo future imaging.