Tracking and mapping the nascent RNA molecules in cells is essential for deciphering embryonic development and neuronal differentiation. Here, we utilized 4-thiouridine (s4U) as a metabolic tag to label nascent RNA and developed a fluorescence imaging method based on the DNA-templated oxidative amination (DTOA) reaction of s4U. The DTOA reaction occurred between amine-modified DNA and s4U-containing RNA with high sequence specificity and chemical selectivity. Target nascent mRNAs in HeLa cells, including those encoding green fluorescent proteins (GFPs) and endogenous BAG-1, were thus lit up selectively by DTOA-based fluorescence in situ hybridization (DTOA FISH). We believe the DTOA conjugation chemistry shown in this study could be generally applied to investigate the spatial distribution of nascent transcription dynamics in cellular processes.