The invention discloses a fluorescent probe, a preparation method thereof and an application of the fluorescent probe for detecting a nucleic acid G-quadruplex structure. The probe is provided with a structure in a general formula (I) is simple and stable in structure and easy to prepare. The probe can be used for specific detection of a nucleic acid G-quadruplex secondary structure, and the G-quadruplex secondary structure in solution can be rapidly detected by a fluorescence spectrometer or direct visual inspection under irradiation of a fluorescent lamp. The probe can be used for detecting the nucleic acid G-quadruplex structure in agarose gel or polyacrylamide gel and can also be used for detecting, marking or displaying existence or distribution of the G-quadruplex structure in living cells. Fluorescent materials have efficient and specific recognition capability for the nucleic acid G-quadruplex structure, have the advantages of excellent cell membrane permeability, low photo-induced toxicity, biological toxicity and photo-bleaching performance and the like, and overcome the shortcomings of high cost, high equipment requirement, complicated technical operation and the like in other detection methods.