The compound, 3',6'-bis(tetramethylethylenedioxyboryl)fluorane, synthesized in this experiment is a kind of H2O2 fluorescence-strengthened reagent, which depends on deprotection mechanism and can examine the H2O2 concentration sensitively. The structure of the compound was confirmed by the IR and H-1 NMR spectra. The influences of reaction time, solvent and base on the probe synthesis were investigated in detail. The response time of the fluorescent probe was also studied and as a result, the optimal time for the H2O2 detection is 3 min. The fluorescence intensity of the probe was gradually increased with the increase of H2O2 concentration in the range of 2.3x10(-12)similar to 2.0x10(-8) mol/L, the detection limit of H2O2 was 2.3x10(-12) mol/L, and the recovery rates in the blood serum were in the range of 93.9%similar to 102.2%.