<p>Fluorescent nucleoside analogues (FNAs) are essential in probing the structure and dynamics of nucleic acids and interactions of protein. To minimize the perturbation in the fluorescent labeled nucleic acid sequences, FNAs perfectly meet the criteria with natural nucleobases with respect to size as well as hydrogen bonding motif. In this review, recent advancement in spectroscopy and microscopy of different classes of FNAs with their photophysical properties, applications, and future challenges are thoroughly discussed. Engineering of different classes of FNAs with improved fluorescence properties are powerful non-perturbing tools for finding nucleic acids structural dynamics with real time binding interaction in physiological condition. Designing these probes to ensure the fluorescent signal is highly specific to the target-binding event and minimizes structural or chemical disruption of the biological system is substantial. Since FNAs are considered as significant tools for fundamental biological research, having enormous applications such as molecular diagnostic and drug discovery, we further demonstrate their selective structural modification for improved fluorescence properties.</p> Graphical Abstract <p></p>