Enzyme-based fluorescence biosensors and their applications in environmental protection, medicine, and industry are described. Biosensors used in environmental protection measure toxicity effects. A chemical compound or group of compounds is detected by the recognition of molecules in the receptor layer and then by detecting a signal passing through the transducer layer. Biosensors are classified according to the transduction method. Special emphasis is placed on optical biosensors, especially fluorescent biosensors, and such measurement techniques as FRET (Fröster resonance energy transfer), FLIM (fluorescence lifetime imaging), FCS (fluorescence correlation spectroscopy), and changes in fluorescence intensity. The phenomenon of fluorescence in biosensors and the selection of appropriate methods are described. The use of enzymes in the receptor layer and enzyme classification according to its category and functions used for analyte detection are presented. The fluorescence properties of enzymes resulting from possessing such cofactors as flavin or heme (prosthetic) groups are discussed. Several methods for enzyme immobilization, namely entrapment, adsorption, covalent immobilization, cross linking, and affinity interaction are described, and the use of enzymatic fluorescence biosensors in the detection of analytes is presented.