The invention relates to the technical field of detection materials, in particular to a near-infrared bio-thiol fluorescent probe as well as a preparation method and an application thereof. The structural formula of the near-infrared bio-thiol fluorescent probe is shown in the description. The preparation method of the near-infrared bio-thiol fluorescent probe comprises the following step: the target compound is prepared from a compound A and 2,4-dinitrobenzenesulfonyl chloride by a reaction, wherein the structural formula of the compound A is shown in the description. The compound almost hasno fluorescence but emits intense fluorescence after a reaction with bio-thiol, the fluorescence emission wavelength is larger than 650 nm and is in the near-infrared region, and the fluorescence canbe distinguished from autofluorescence of body tissue and can effectively penetrate the tissue to eliminate biological background interference. The compound can be used for detecting bio-thiol, is simple to operate, has fast and sensitive double-response and has broad application prospects in the fields of biomolecular detection and environment.