A new application of ionic liquids in the preparation of fluorescence probe 5,5-bis-8-phenylamino-l-naphthylsulfonate (bis-ANS) is represented. The method for the preparation of fluorescence probe bis-ANS in alkyl imidazolinm cationic liquids under acidic conditions is described. The effects on reaction yields under different concentration of sodium nitrite and different ionic liquid were studied and good yields were achieved.