The polarization of light emitted by fluorescent probes is an easily accessible physical quantity that is related to a multitude of molecular parameters including conformation, orientation, size and the nanoscale environment conditions, such as dynamic viscosity and temperature. In analytical biochemistry and analytical chemistry applied to biological problems, fluorescence anisotropy is widely used for measuring the folding state of proteins and nucleic acids, and the affinity constant of ligands through titration experiments. The emphasis of this review is on new multi-parameter single-molecule detection schemes and their bioanalytical applications, and on the use of ensemble polarization assays to study binding and conformational dynamics of proteins and aptamers and for high-throughput discovery of small-molecule drugs.