<p>Polymethine dyes (PDs) possessing unique photophysical and photochemical properties are widely used as probes or sensors for studying biomolecular systems. This review systematizes the results of our studies of the interaction between PDs and biomolecules (DNA, albumins, etc.) as part of research of certain biological objects. The fluorescent and photochemical properties of PDs are considered and the potential of application of these compounds as probes/sensors for biomolecular systems is assessed. In particular, interactions of cationic PDs with DNA and other biomolecules and those of anionic and cationic PDs with proteins, as well as the applicability of PDs as sensors in biomicellar systems and the application of molecular docking in studies of the interaction of PDs with biomolecules are discussed. It is demonstrated that improvement of PD probes based on deeper understanding of structure–property relationships, the development of specialized sensors for solving various biomedical problems, and further investigations of elementary photochemical processes occurring in the PD complexes with biomolecules still remain key areas of the development.</p>