Abstract Fluorescent nucleoside analogues (FNAs) are indispensable in studying the interactions of nucleic acids with nucleic acid-binding proteins. By replacing one of the poorly emissive natural nucleosides, FNAs enable real-time optical monitoring of the binding interactions in solutions, under physiologically relevant conditions, with high sensitivity. Besides that, FNAs are widely used to probe conformational dynamics of biomolecular complexes using time-resolved fluorescence methods. Because of that, FNAs are tools of high utility for fundamental biological research, with potential applications in molecular diagnostics and drug discovery. Here I review the structural and physical factors that can be used for the conversion of the molecular binding events into a detectable fluorescence output. Typical environmentally sensitive FNAs, their properties and applications, and future challenges in the field are discussed.