MicroRNAs (miRNAs) are small noncoding RNAs that play important regulatory roles in multiple biological processes. Many miRNAs exhibit unique expression patterns and are considered as theranostic biomarkers in a variety of human diseases. A reporter system that is capable of imaging miRNA in vivo is crucial for investigating miRNA biology. In the present study, an organic anion-transporting polypeptide 1B3 (OATP1B3)-based genetic switch system is designed and optimized to achieve near-infrared fluorescent imaging of miRNA by the uptake of indocyanine green (ICG) dye. The reporter system, named miR-ON-OB3, is shown to be efficient to regulate the expression of OATP1B3 in mammalian cells. Notably, the results indicate that the system is of high sensitivity for near-infrared fluorescence imaging of both exogenous and endogenous miRNA in mammalian cells. Moreover, the system is proved to be functional for real-time near-infrared fluorescence imaging of miRNA in living mice. This study establishes a novel genetic encoded reporter for near-infrared fluorescence imaging of miRNA, which may provide a potential tool for in vivo imaging of miRNA in clinical applications due to the clinical availability of ICG.