A detailed protocol for the synthesis of a fluorescent pyrimidine ribonucleoside analogue and its enzymatic incorporation into an RNA strand by transcription reactions is described. Furan-modified ribonucleoside triphosphate is synthesized in two steps with an overall yield of 33%. Incorporation of the triphosphate into an RNA oligomer occurs with nearly 225-fold amplification over the amount of the DNA template. Bacterial rRNA decoding site (known as the A-site) derived from this fluorescently modified ssRNA positively signals a binding event upon interaction with aminoglycoside antibiotics, its cognate ligands. The total time for the synthesis of ribonucleoside triphosphate is ∼6 days, and that for the incorporation of the nucleoside triphosphate and purification of the fluorescently labeled RNA ∼40 h.