Antisense oligonucleotides have been used for a variety of purposes in microRNA (miRNA) research including functional evaluation, target recognition, and gene studies. Although several (99m)Tc-radiolabeled oligonucleotides have been reported in antisense imaging, none of those were related to miRNA tracing. Moreover, separation after labeling was always required to achieve acceptable radiochemical purity. In this study, we prepared a (99m)Tc-radiolabeled oligonucleotide under optimized conditions for the purpose of tracing miRNA. A 22mer anti-miRNA oligonucleotide (AMO) was designed completely complementary to the sequence of mature miR-21. AMO probe modified with 2'-O-Methyl and phosphorothioate backbone was designed and synthesized. This probe was conjugated with a bifunctional chelator S-acetylmercaptoacetyltriglycine (NHS-MAG3) via a primary amine on 5'-end. Furthermore, it was radiolabeled and its optimization labeling conditions were performed by varying the amount of stannous ion, (99m)Tc-pertecnetate, and reaction time, respectively. Finally, the labeled product was identified by gel electrophoresis and evaluated for its serum stability. The AMO was synthesized with partial 2'-OMe and phosphorothioate modification to improve its stability. Excess of MAG3 impurity was removed by precipitation of tin and MAG3 after the conjugation. The labeling efficiency reached 97 % under the optimal reaction conditions of 2 μg/μL SnCl2·2H2O addition, (99m)Tc solution with high specific activity, and 90-min reaction at room temperature. Gel electrophoresis confirmed that the peak of radioactivity located the same position of oligomer, which identified the successful radiolabeling. After incubated with human fresh serum for 12 h, labeled AMO showed good stability with high radiochemical purity and no significant degradation. A (99m)Tc-labeled AMO targeting miR-21 can be prepared with high labeling efficiency under optimized conditions, which provides a good support for the future use of miRNA-targeted tracing and imaging.