As one vital member among the family of phosphates, guanosine triphosphate (GTP) not only plays a very important role in many critical biological processes but also closely associates with definite pathological states. Based on the ratiometric fluorescence response of the zinc complex of 1,4-bis(imidazol-1-ylmethyl) benzene (bix) in this contribution, a highly selective recognition of GTP has been successfully developed. The fluorescence of bix-Zn(II) at 289 nm decreased in the presence of GTP with the appearance of one new emission band at 341 nm, resulting in ratiometric fluorescence changes with the concentration of GTP. With that, ratiometric fluorescence recognition for GTP could be effectively established, and so GTP could be successfully discriminated from other structurally similar anions, including ATP and PPi. Furthermore, bix-Zn(II) also has a ratiometric fluorescence response to DNA sequences containing guanine.