The use of fluorescent nucleic acid base analogues is becoming increasingly important in the fields of biology, biochemistry and biophysical chemistry as well as in the field of DNA nanotechnology. The advantage of being able to incorporate a fluorescent probe molecule close to the site of examination in the nucleic acid-containing system of interest with merely a minimal perturbation to the natural structure makes fluorescent base analogues highly attractive. In recent years, there has been a growing interest in developing novel candidates in this group of fluorophores for utilization in various investigations. This review describes the different classes of fluorophores that can be used for studying nucleic acid-containing systems, with an emphasis on choosing the right kind of probe for the system under investigation. It describes the characteristics of the large group of base analogues that has an emission that is sensitive to the surrounding microenvironment and gives examples of investigations in which this group of molecules has been used so far. Furthermore, the characterization and use of fluorescent base analogues that are virtually insensitive to changes in their microenvironment are described in detail. This group of base analogues can be used in several fluorescence investigations of nucleic acids, especially in fluorescence anisotropy and fluorescence resonance energy transfer (FRET) measurements. Finally, the development and characterization of the first nucleic base analogue FRET pair, tC(O)-tC(nitro), and its possible future uses are discussed.