A method based on capillary electrophoresis coupled to laser-induced fluorescence detection was developed for the characterization of fatty acids including palmitic, stearic, oleic and tuberculostearic acids. The fatty acids were tagged by 4-aminofluorescein (AF) via a carboxylic acid-amine condensation promoted by N'-(3-dimethylaminopropyl)-N-ethylcarbodiimide (EDC) in non-aqueous solution. Using the optimized derivatization conditions, the fluorophore labeling of the fatty acids was achieved at the nanomolar level. The separation of palmitic, stearic, oleic and tuberculostearic-AF derivatives was achieved in less than 10 min, using 25 mM sodium borate buffer containing 30% of acetonitrile as running electrolyte. The concentration detection limit was found to be 5 nM while the minimum mass limit detection is around 30 attomol. This method was successfully applied to identification of mycobacteria via the characterization of tuberculostearic acid and found to be suitable for the detection of a minimum of 10(6) mycobacteria.