The invention discloses a fluorine-boron fluorescent dye as well as a preparation method and application thereof, wherein the structure of the fluorine-boron fluorescent dye is shown as a formula (I) or a formula (II), in the formula (I) and the formula (II), R1 is H or halogen; R2 is CN; R3, R4, R5 and R6 are independently selected from H, halogen, C1-C6 alkyl or C1-C6 alkoxy; V, W, X, Y and Z are independently CH or N, and when V, W, X, Y or Z is N, N has no substituent group. According to the fluorine-boron fluorescent dye and the preparation method thereof, the maximal fluorescence emission wavelength of the fluorine-boron fluorescent dye is 518-600nm, and the fluorine-boron fluorescent dye also has excellent fluorescence quantum yield and Stokes shift, which shows that the fluorine-boron fluorescent dye has good application prospect in the bioanalysis fields of fluorescence labeling, bioimaging and so on; meanwhile, the preparation method is simple in steps, and raw materials can be obtained easily.