The invention discloses a rhodamine B-based fluorescent sensor, preparation and application thereof. 2-(2-aminoethyl)-3',6'-bis(diethyl-amino)spiro[isoindoline-1,9'-xanthen]-3-one(A) and 4-(1,4,7,10-tetraoxa-13-azacycl-opentadecan-13-yl)-4-oxobutanoicacid(B) are taken as precursors to synthesize a fluorescent sensor (C). After addition of different heavy metal ions like NaCl, LiCl.H2O, KCl, NiCl, CuCl, AgNO3, SnCl2.2H2O, Ba(NO3)2, MnCl2.4H2O, ZnCl12, MgCl2.6H2O, CaCl2, CdCl2.2.5H2O, CuCl2.2H2O, FeCl2.4H2O, PbCl2, CoCl2.6H2O, HgCl2, Pd(C2H3O2)2, RhCl3.H2O, IrCl3, AlCl3, FeCl3.6H2O, and CrCl3.6H2O, analysis by ultraviolet-visible spectrum and spectrofluorimetry finds that the sensor only responses to iron ions, so that the fluorescent sensor can be used for specially detecting iron ions. The sensor has advantages of high sensitivity, simple operation and good specificity, and has important potential significance in disease diagnosis.