Amphotericin B (AmB) is a polyene antibiotic used to treat deep-seated mycoses. Both the pharmaceutical and toxic activities of AmB depend on the molecular organization of the drug. The fluorescence of AmB has proven to be a powerful technique of studying the drug's association state. In particular, fluorescence lifetime appeared to be sensitive to the formation of AmB dimers and aggregated structures. This paper addresses the application of the fluorescence technique in the study of the molecular organization of AmB, and perspectives on future application of this approach are addressed briefly.