The invention discloses a double-excitation fluorescence probe and a preparation method and application thereof in detection of hydrazine in water. The preparation method includes: taking diethylenetriamine pentaacetic acid, acetic anhydride and pyridine, stirring and heating for 24h at 65 DEG C, cooling, filtering, washing and drying; well mixing obtained diethylenetriamine pentaacetic acid dianhydride with triethylamine, DMF (dimethyl formamide) and fluorescein, heating and stirring for 24h at a constant temperature of 100 DEG C, cooling, filtering, and drying; dissolving obtained diethylenetriamine pentaacetic acid-bis(fluorescein) into Tris-HCl/DMSO (10mmol.L<-1>, v/v=9:1) buffer solution with pH equal to 7.5, mixing with Tb(NO3)3.6H2O, and heating or placing for more than two hours toobtain a target product. Uric acid detection is realized by combination of Tb<3+>-dtpa-bis(fluorescein) as a double-excitation probe and a fluorescence method. The method is simple, novel, high in efficiency and low in cost, and applicability to practical water samples is realized.