Rhamnolipids (RLs) are a class of glycolipid produced by Pseudomonas aeruginosa, frequently cited as the best characterized surfactants micro-organism. RLs are composed of one or two rhamnoses conjugated to fatty acid chains. RLs possess unique high surface activity and thus have been extensively gained interests in various industrial applications as emulsifiers, detergents and antibiotics etc. As new promising biotechnological products, RLs are the most investigated biosurfactants due to their commercial availability. Over the past years, great efforts have been achieved and a collection of techniques has been developed for the isolation of producing bacterial strains and the characterization of a large variety of rhamnolipids homologs and congeners. Infrared (IR) spectroscopy and electrospray ionization mass spectrometry (ESI–MS) can be used for the identification of the isolated rhamnolipids. The results of IR spectra confirmed that the isolated compounds corresponded to the rhamnolipids structure, whereas MS indicated that the isolated preparation was a combination of mono-rhamno-mono-lipidic, mono-rhamno-di-lipidic and di-rhamno-di-lipidic congeners. Rhamnolipids can be potentially applied in many fields from bioremediation to food additives and several additional applications. This review is focused on the production of RLs, the different techniques of RLs identification, and the applications of RLs in food industry and agriculture