Objective： MicroRNA-155 (miR-155) is significantly highly expressed in breast cancer, lung cancer, liver cancer and other malignant tumors. This study was to design and construct a radiolabeled probe targeting miR-155 for in vivo imaging in breast cancer. Methods: Anti-miR-155 oligonu-cleotide (AMO-155) was chemically synthesized with 2′-OMe modification. Its 5′ end was linked with acetyl amine group. After chelated with a bifunctional chelator NHSMAG3, AMO155 was radiolabeled with 99mTc using stannous chloride. The serum stability was evaluated at cellular level. In vivo imaging was performed in MCF-7 tumor bearing mice after the administration of 99mTc radiolabeled AMO-155 and scramble control probes, respectively. Furthermore, the blocked imaging of tumor bearing mice was obtained after the injection of unlabeled AMO-155 2 hours ahead. MCF-7 and MDA-MB-231 tumor bearing mice with different expression level of miR-155 were imaged, respectively. Quantitative real-time PCR (qRT-PCR) was used to identify the expression level of miR-155 in the bearing tumors. Results: 99mTc-AMO-155 was prepared with high radiolabeled efficiency (97%), radiochemical purity (greater than 98%), and radioactive specific activity (3.75 GBq/μg). 99mTc-AMO-155 was stable in fresh human serum for 12 hours. After the administration via tail vein, 99mTc-AMO-155 displayed significant accumulation in MCF-7 bearing tumors with high expression level of miR-155, whereas 99mTc-control showed little accumulation. After blocked with unlabeled AMO-155, the tumor could not be visualized clearly after the administration of 99mTc-AMO-155. Furthermore, 99mTc-AMO-155 could show the differential expression of miR-155 in vivo. MCF-7 tumor was shown with significantly higher radioactive accumulation than MDA-MB-231, based on its higher expression level of miR-155, which was verified by qRT-PCR. Conclusion: 99mTc-labeled AMO-155 with chemical modification showed good serum stability and in vivo tumor targeting ability. This study provides a potential probe for in vivo imaging of breast cancer.