Nine fluorescent 5′–6-locked nucleosides were synthesized by condensation of various 1,2-diketones with 5-amino-2′-deoxycytidine. The nucleosides have different substituents on the pyrazine core structure, ranging from two methyl groups to polyaromatic rings. The photophysical properties of each nucleoside were determined, with the nucleosides displaying diverse absorption and emission maxima, extinction coefficients and quantum yields. The nucleoside with the highest fluorescence brightness was phosphitylated and incorporated into an oligonucleotide by means of automated oligonucleotide synthesis. The labelled oligonucleotide in aqueous buffer exhibited a substantially lowered extinction coefficient and quantum yield compared to the nucleoside in THF. The photophysical properties of the nucleoside were also compared in different DNA structural contexts, a single strand, a 14-mer duplex, a 14-mer duplex with an 11-mer overhang, and a 25-mer nicked duplex labelled at the nick site. Circular dichroism and melting temperature studies verified that the nucleoside did not perturb or destabilize the DNA helixes. In fact, when incorporated at the nick site, the nucleoside was found to stabilize the nicked duplex notably compared to its unmodified counterpart. The brightness of the fluorescent nucleoside in DNA increased as the polarity of its surroundings decreased, being highest in the 25-mer nicked duplex where exposure to the polar solvent is minimized by stacking to the adjacent bases on both the 3′- and 5′-side. The nucleosides brightness in the nicked duplex was also found to increase with lowered temperature, in accordance with expected temperature-dependent changes in the stacked–unstacked equilibrium at the nick site.